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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ricin A-chain was used as a test protein to study the effects of deletion of codons on the ribosomal synthesis, release and chaperone-mediated folding of the proteins. Synthesis of wild-type ricin and five mutant proteins was carried out in an Escherichia coli cell-free coupled transcription/translation system from otherwise identical non-linearized plasmids. The deletions involved small numbers of contiguous amino acid residues at different points from the N terminus to the C terminus of the wild-type protein. Deletion of the N-terminal 20 amino acid residues caused a 45% reduction in total protein synthesis whereas deletion of the next three amino acid residues caused a 1.5-fold increase in synthesis compared with wild-type with an accumulation of full-length polypeptides as peptidyl-tRNA in the ribosomal P site. Intermediate levels of synthesis and release were seen with the other three mutants. Enzymatic activity was detected only with wild-type protein and a mutant lacking the C-terminal five amino acid residues. These were the only ricin species in which chaperone-dependent reactions could be detected by fluorescence from coumarin incorporated with methionine at the N terminus of the proteins. By using sparsomycin to block termination of full-length peptidyl-tRNA, it was demonstrated that the chaperone-mediated reactions detected by fluorescence occur on the ribosomes and involve folding of the nascent protein as peptidyl-tRNA. The results presented provide a direct demonstration of two points of fundamental importance: folding of nascent proteins involving chaperone-mediated reactions can occur on ribosomes and is directly related to the conformation of the native enzyme. Deletion of amino acid residues at different points from the N terminus to the C terminus affects the reactions of elongation, chaperone-mediated folding and release of full-length protein.
J Mol Biol 1995 Sep 15
PMID:Elongation and folding of nascent ricin chains as peptidyl-tRNA on ribosomes: the effect of amino acid deletions on these processes. 767 1

Since, linking of ovine luteinizing hormone (oLH) to ribosome inactivating protein gelonin (in oLH-gelonin conjugate) occur via the alpha-subunit, alpha oLH, an attempt has been made to develop a 'universal' hormonotoxin for selective targeting to specific cells in the gonads. Four different molar ratios of oLH and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) were used to activate the epsilon amino (phi-NH2) groups of alpha oLH. The alpha oLH-SPDP derivatives recombine to native beta subunit of oLH (beta oLH) and the purified recombinants retained substantial receptor binding, steroidogenic activity and immunoreactivity to native oLH. The disulfide linked alpha oLH-S-S-gelonin conjugates prepared by SPDP method were purified by gel filtration chromatography and analysed by reverse-phase high performance liquid chromatography (RP-HPLC). In order to obtain specificity and bioeffectivity, the alpha oLH-S-S-gelonin conjugates were allowed to recombine to native beta oLH and the recombination mixture was further purified by gel-filtration chromatography. The RP-HPLC analysis of these recombinants indicated that alpha oLH-S-S-gelonin did not recombine to beta oLH. The failure of recombination may be due to the reasons. (i) The site of epsilon-NH2 activation by SPDP may be different in the alpha oLH than the native oLH. (ii) The activation site may be in close proximity to the annealing site which facilitates the recombination of beta-subunit but failed to reassociate to alpha oLH-S-S-gelonin conjugate.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1993 Mar 24
PMID:Disulfide linked alpha oLH-gelonin conjugate failed to recombine with beta oLH subunit to generate bioeffective hormonotoxin. 768 46

Escherichia coli cells transformed with plasmids containing ricin B-chain coding sequences are shown to express this heterologous protein in inclusion bodies. After denaturation and renaturation of the product in the presence of glutathione and lactose, the recombinant ricin B-chain is soluble, biologically active and stable. Cytotoxicity of heterodimer containing this protein and ricin A-chain is found to be only ten times lower, than that of native ricin. Recombinant B-chain alone was nontoxic to cells (ID50 > 10(-6) M). Our data suggest that ricin B-chain oligosaccharides are essential for stability preserving protein from proteolytic degradation in cells.
Mol Biol (Mosk)
PMID:[Production of biologically active recombinant ricin B-chain]. 778 43

Alkaline (pI 8.6-7.5) and neutral (pI 7.0-6.0) isoforms of human TSH have been isolated from a highly purified intrapituitary preparation by isoelectric focusing and compared for their respective actions on thyroid cell proliferation. Both TSH isoforms displayed the same ability to bind to porcine thyroid membranes as the original hormone preparation, indicating a similar recognition at the receptor sites. Alkaline forms showed a higher potency in inducing either cyclic AMP (cAMP) production or [3H]thymidine incorporation in FRTL-5 cells (half-maximal effective doses (ED50 values) = 0.25 and 0.29 nM respectively) compared with their neutral counterparts (ED50 values = 0.66 and 0.70 nM respectively). Increasing the concentration of alkaline forms in the presence of a half-maximal concentration of neutral TSH resulted in a profound inhibition of cell growth without a significant change in cAMP. Conversely, increasing the amount of neutral forms in the presence of a half-maximal dose of alkaline TSH resulted in an additive response for cAMP production but not in cell proliferation. To assess whether glycosylation might be responsible for the variation in hormone action, both alkaline and neutral TSH isoforms were tested for recognition of their carbohydrate chains by concanavalin A (Con A) and ricin. No major difference was found in binding to Con A, indicating that the contribution of carbohydrates to changes in hormone pI was not related to core branching. Very few galactose residues were accessible in either hormone fraction since little binding to ricin was observed. Isoelectric focusing of TSH forms before and after neuraminidase treatment revealed that neutral forms had a higher sialic acid content than alkaline TSH. In conclusion, the current findings show that TSH isoforms differentially affect cAMP production and cell growth. TSH fractions with a high sialic acid content and a low mitogenic activity behave as antagonists to the more active forms for cell proliferation. It is suggested that physiological control of TSH action at the thyroid gland may reside in the respective amounts of various TSH forms which, once bound to their receptor, can induce variable activation of post-receptor events while controlling cell proliferation.
J Mol Endocrinol 1994 Oct
PMID:Dual activity of human pituitary thyrotrophin isoforms on thyroid cell growth. 784 30

The conformation of a 29 base oligonucleotide called E73 has been determined in solution by NMR. E73 includes a 23 nucleotide sequence that is identical with that of a the alpha-sarcin and ricin-sensitive loop (SRL) from rat 28 S rRNA, and like the SRL in intact ribosomes, E73 is a substrate for both toxins. The SRL includes a long, conserved sequence found in the RNA of all large ribosomal subunits, which plays a critical role in the factor-dependent steps of protein synthesis. The spectroscopic observations and analysis that led to the determination of the conformation of E73 are presented. The SRL in E73 has a highly structured conformation, which is stabilized by several non-Watson-Crick base-pairs, and many properties of the SRL in the ribosome can be understood assuming that the conformation of E73 and that of the SRL in the ribosome are the same. The role of the SRL in protein synthesis is discussed in light of the conformation of E73, as is the modular relationship that exists between the conformation of the SRL and other smaller RNAs.
J Mol Biol 1995 Mar 17
PMID:The sarcin/ricin loop, a modular RNA. 789 62

To investigate whether glycanic chains of prolactin receptors (PRL-R) play a role in hormone binding activity, comparison was made of rat and mouse liver solubilized receptors with respect to both their affinity for the hormone and their glycosylation properties. As compared with rat receptors, mouse receptors exhibited a 2-fold higher affinity for human growth hormone (hGH), the hormone being bound by both tissues with a lactogenic specificity. Along with this increased affinity, mouse receptors had a 2 lower M(r) relative to rat receptors (62 kDa versus 64 kDa as measured on hGH cross-linked receptors). These differences could be ascribed to different glycosylation properties of the receptors from the two species, as supported by the followings. 1) After treatment with endoglycosidase F (endo F), rat and mouse PRL-R no longer exhibited any difference in their M(r) (54 kDa for both cross-linked receptors). 2) Neuraminidase treatment increased by 37% the binding of hGH to mouse receptors, but was ineffective on the hormone-binding to rat receptors. Conversely, wheat germ agglutinin (WGA), another sialic acid specific probe, decreased hGH binding to rat receptors by 25%, but had no effect on this process for mouse ones. 3) Marked differences were observed in the recoveries of rat and mouse hormone-receptor (HR) complexes from ricin-1- (RCA1-), concanavalin A- (ConA-) and WGA-immobilized lectins. These differences were reduced (RCA1 and ConA) or abolished (WGA) after rat and mouse receptor desialylation by neuraminidase, a treatment which decreased the M(r) of both receptors by 2 kDa. Taken together, these results strongly suggest that the PRL-R from rat and mouse liver contain biantennary N-linked oligosaccharidic chains with distinct type of sialylation, which may account for their differential hormone-binding affinities.
Cell Mol Biol (Noisy-le-grand) 1994 May
PMID:Differences in both glycosylation and binding properties between rat and mouse liver prolactin receptors. 792 Jan 80

We have used chemical modification analysis to probe the solution structure of the hairpin ribozyme. The modifying reagents dimethylsulfate, 1-cyclohexyl-N'-[2-(N-methylmorpholino) ethyl-carbodiimide-p-toluenesulfonate, kethoxal, diethylpyrocarbonate and (2,12-dimethyl-3,7,11,17- tetraazabicyclo [11.3.1]heptadeca-1(17),2,11,13,15-pentaenato) nickel(II) perchlorate were used to probe functional groups that participate in Watson-Crick and non-canonical base-pairs. Our results confirm the existence of four short helices (3 to 6 bp) within the ribozyme-substrate complex, and demonstrate that one intramolecular helix (helix 4) is comprised of three base-pairs rather than the previously suggested five. In the absence of magnesium, the ribozyme is observed to fold into its secondary structure. Upon addition of magnesium, a striking difference in chemical modification is observed, particularly at sites within the ribozyme's large internal loop (loop B) that are essential for catalytic function (bases 21 to 26). Moreover, magnesium-dependent folding clearly destabilizes an A-U base-pair in a region where a proposed bend is required to juxtapose the catalytically essential loops A and B. Upon addition of substrate, no changes are observed in the structure of helix 3, loop B or helix 4. However, strong protection of bases in the substrate-binding domain is observed, including those located across internal loop A. The modification data are consistent with the formation of a previously proposed tertiary structure motif within loop B that includes non-canonical G-A, A-U and A-A base-pairs, and that is identical with those identified by NMR analysis of loop E of 5 S rRNA and the sarcin/ricin loop of 28 S rRNA. Our results indicate that the hairpin ribozyme adopts a stable magnesium-dependent tertiary structure to which the substrate binds without inducing major conformational changes, and that substrate recognition is likely to involve non-canonical base-pairs between the ribozyme and substrate sequences adjacent to the cleavage site.
J Mol Biol 1994 Nov 18
PMID:Structure-mapping of the hairpin ribozyme. Magnesium-dependent folding and evidence for tertiary interactions within the ribozyme-substrate complex. 796 21

Ricin is a potent plant toxin which acts by removing a specific adenine residue from the ribosome. The X-ray crystal structure of a new, tetragonal crystal form of the recombinant ricin A-chain diffracting to 1.8 A resolution has been determined via molecular replacement methods and refined to a crystallographic R-factor of 18.6%. The higher resolution electron density allowed improvements to be made upon previously published models, resulting in an increase in the assigned secondary structure of the protein. The enzyme adopts the same global conformation in this crystal form with differences in detail due only partly to crystal packing. The active site superimposes closely with those of previously published models but the locations of the active-site water molecules differ in this structure. To address the current mechanistic model, an additional two structures are presented: recombinant ricin A-chain complexed with the substrate analogue formycin monophosphate as well as with adenosine monophosphate, which is cleaved by the crystalline enzyme. The formycin monophosphate displaces a putative catalytic water molecule. This supports the notion that the analogue does not bind in a transition state conformation and that contacts from other elements of the 28 S RNA natural substrate are required to achieve full reactivity. The structure of the adenosine monophosphate complex suggests a mechanism for the release of the adenine product via of the side-chain Tyr80. The structures suggest that Glu177 is better positioned for the activation of the catalytic water molecule than Arg180.
J Mol Biol 1994 Dec 09
PMID:X-ray structure of recombinant ricin A-chain at 1.8 A resolution. 799 Jan 30

Escherichia coli cells transformed with plasmids containing ricin B-chain coding sequences are shown to express this heterologous protein in inclusion bodies. After denaturation and renaturation of the product in the presence of glutathione and lactose, the recombinant ricin B-chain is soluble, biologically active and stable. Cytotoxicity of heterodimer with this protein and ricin A-chain is bound to be only ten times less than of native ricin. Recombinant B-chain alone was nontoxic to cells (ID50 > 10(-6)M). Our data suggest that N-glycosylation of ricin B-chain is not required for its biological activity.
Biochem Mol Biol Int 1994 Apr
PMID:Renaturated ricin toxin B chain made in Escherichia coli is soluble, stable, and biologically active. 806 31

The immunological relatedness of various ribosome-inactivating proteins (RIPs) from the Cucurbitaceae family was examined using enzyme-linked immunosorbent assay (ELISA). The proteins studied included trichosanthin (TCS), beta-trichosanthin (beta-TCS), alpha-momorcharin (alpha-MMC), beta-momorcharin (beta-MMC), momorcochin (MCC), luffaculin (LFC) and Mirabilis antiviral protein (MAP). In general, an antigen exhibited the highest immunoreactivity in an assay using it own antibodies when compared with others RIPs, and MAP demonstrated the lowest immunoreactivity in most ELISAs employing antibodies raised against the other RIPs. LFC also exhibited very low immunoreactivity in most assays, TCS and beta-TCS are immunologically disparate in a number of ELISAs. alpha-MMC and beta-MMC are immunologically related but not identical. MCC manifested immunological relatedness to the momorcharins to a certain extent.
Biochem Mol Biol Int 1993 Nov
PMID:Immunological relatedness of ribosome-inactivating proteins from the Cucurbitaceae family. 811 19


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