Modification of glucoamylases from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.
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To investigate the role of carboxyl groups of glucoamylases [EC 3.2.1.3] from a Rhizopus sp. (Gluc1 and Gluc2), the modification of Gluc1 and Gluc2 with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), was studied. The inactivation of Gluc1 proceeded with the incorporation of about 3 CMC moieties. In the presence of maltose, the modification of about 2.2 carboxyl groups of Gluc1 proceeded with a slight loss of enzymatic activity. In the re-modification of Gluc1 modified in the presence of maltose, Gluc1 was inactivated by further modification of about 1.3 carboxyl groups. Therefore, one carboxyl group, which was protected by maltose, was thought to be a crucial one. The inactivation of Gluc2 proceeded similarly to that of Gluc1, but the number of CMC moieties incorporated was about one less than in the case of Gluc1. Thus, it was suggested that one of the reactive carboxyl groups of Gluc1 was located in the N-terminal part of Gluc1, which is deficient in Gluc2. From the results of kinetic studies on CMC-modified Gluc1, it was suggested that the hydrolysis mechanism of malto-oligomers differs somewhat from that of PNPG. (+info)
Modification of a major ribonuclease from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.
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In order to investigate the role of carboxyl groups of a base non-specific ribonuclease from Aspergillus saitoi [EC 3.1.27.1] (RNase M, molecular weight 36,000), the modification of RNase M with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide(CMC), was studied. The inactivation of RNase M proceeded almost linearly with the incorporation of about 9.5 CMC moieties. The peptide backbone structure of the modified RNase M was practically the same as that of the native RNase M, as assessed from the CD spectra in the region of 200-250 nm. In the presence of competitive inhibitors, adenosine, and cytidine, inactivation of RNase M by CMC was partially inhibited. In the presence of cytidine (1 M), the modification of about 4 carboxyl groups of RNase M proceeded with a slight loss of enzymatic activity (ca. 20%). Further modification inactivated RNase M with the incorporation of ca. 4-5 CMC without any detectable intramolecular peptide bond formation. Therefore, it was concluded that carboxyl groups responsible for enzymatic activity were included among these carboxyl groups protected by cytidine. The logarithm of the half-live of the inactivation of RNase M by CMC was a linear function of log[CMC] with a slope of minus one, indicating that at least one carboxyl group among the modified ones may be essential for catalysis. The digestion of CMC-modified RNase M with carboxypeptidase A eliminated the carboxyl terminal group from the site of CMC modification. (+info)
Modification of a glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.
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1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4)-glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with water-soluble carbodiimides was studied. 2. Gluc M1 was inactivated most effectively by 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide (CMC) at pH 4.5. 3. Inactivation of Gluc M1 with [14C]CMC proceeded with the incorporation of about 12 CMC moieties. From the results of amino acid analysis, titration of SH group with Ellman's reagent and hydroxylamine treatment at pH 7.0, it was concluded that the crucial sites of modification were carboxyl groups of Gluc M1. 4. The CD spectrum of CMC-modified Gluc M1 (residual activity, ca. 9.8%) suggested that the gross conformation of the native enzyme was retained. 5. In the presence of maltose, when Gluc M1 was incubated with [14C]CMC, ca. 10 CMC moieties were incorporated with a simultaneous decrease in enzymatic activity (30%). The Gluc M1 modified in the presence of maltose was remodified with CMC after elimination of maltose. The CMC-modified Gluc M1 was inactivated completely with the incorporation of ca. 4 CMC moieties. 6. The logarithm of the half-life of the inactivation of Gluc M1 by CMC was a linear function of log [CMC] indicating that one carboxyl group among the modified ones was crucial for inactivation of Gluc M1. 7. The protection by maltose of Gluc M1 from inactivation and the increase in K1 values for maltose of CMC-modified Gluc M1's suggested that a crucial carboxyl group(s) was located near or on subsites 2 and 3. (+info)
Modification of carboxyl groups in Geotrichum candidum lipase.
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Lipase from Geotrichum (Geo.) candidum was rapidly inactivated by incubation with water-soluble carbodiimide, 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC), or 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl) carbodiimide metho-p-toluenesulfonate (CMC), at pH 4.8. The pH dependence of the rate of inactivation was consistent with the modification of carboxyl groups in the lipase. Reaction of the lipase with EDC in the presence of the nucleophile taurine showed that about 9 carboxyl groups per molecule of enzyme were modified with concomitant total loss of activity. This number was reduced to 4 when CMC was used as a carbodiimide instead of EDC. The modification had no effect on the CD spectrum in the ultraviolet region. Kinetic analysis of the effect of CMC on the lipase indicated that at least 1 CMC molecule bound to the enzyme during inactivation. (+info)
Carbodiimide modification analysis of aminoacylated yeast phenylalanine tRNA: evidence for change in the apex region.
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The G- and U-specific reagent, carbodiimide was used to probe the solution structure of aminoacylated yeast phenylalanine tRNA. Both quantitative and qualitative changes in modification were observed when the modification patterns of tRNA-CCA(3'OH), tRNA-CCA(3'NH2) and phe-tRNA-CCA(3'NH2) were compared. Five nucleotides were modified in all cases, D16 and G20 in the D-loop, U33 and Gm34 in the anticodon loop and U47, in the region of the extra arm. Small changes occurred in the D-loop with incorporation of the adenosine analogue manifest as new, low levels of modification of G22 (D-stem) and a loss of sensitivity to Mg+2 in modification of D16. Aminoacylation resulted in new modification of G19, modification of a residue in the T psi CG sequence, and a 2.5-fold increase in modification of G22. Taken together the results show that aminoacylation causes increased exposure of bases in the apex region of the L-shaped molecule where the D- and psi-loops are joined. The effects observed could occur as a consequence of stable or dynamic changes in conformation. (+info)
In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations.
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The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. Previous in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate conformations, Form 1 and Form 2, with Form 1 being the dominant one. To gain information about the structure of the SL RNA in vivo, in its protein-rich environment, we have used permeable Trypanosoma brucei and L. collosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contrast to the in vitro results, the Form 2 secondary structure predominates. However, there are chemically accessible regions that suggest conformational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary interactions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. SL RNA stripped of proteins and probed in vitro does not display the same Form 2 bias, indicating that SL RNA structure is determined, at least in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vivo. (+info)
Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ.
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The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification. (+info)
Solution structure of mRNA hairpins promoting selenocysteine incorporation in Escherichia coli and their base-specific interaction with special elongation factor SELB.
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On the basis of chemical probing data, the solution structures of RNA hairpins within fdhF and fdnG mRNAs in Escherichia coli, which both promote selenocysteine incorporation at UGA codons, were derived with the help of computer modeling. We find that these mRNA hairpins contain two separate structural domains that possibly also exert two different functions. The first domain is comprised of the UGA codon, which is included within a complex and distorted double-stranded region. Thereby, release factor 2 might be prevented from binding to the UGA codon to terminate protein synthesis. The second domain is located within the apical loop of the mRNA hairpin structures. This loop region exhibits a defined tertiary structure in which no base is involved in Watson-Crick interactions. The structure of the loop is such that, following a sharp turn after G22 (A22 in fdnG mRNA), bases G23 and U24 are exposed to the solvent on the deep groove side of the supporting helix. Residues C25 and U26 close the loop with a possible single H-bonding interaction between the first and last residues of the loop, 04(U26) and N6(A21). The bulge residues U17 and U18 (in fdhF mRNA), or Ul7 only in fdnG mRNA, point their Watson-Crick positions in the same direction as loop residues G23 and U24 do, and at the same time open up the deep groove at the top of the hairpin helix. Chemical probing data demonstrate that bases G23 and U24 in both mRNA hairpins, as well as residues U17 and Ul7/U18 (for fdhF mRNA) located in a bulge 5' to the loop, are involved directly in binding to special elongation factor SELB in both mRNAs. Therefore, SELB recognizes identical bases within both mRNA hairpins despite differences in their primary sequence, consistent with the derived 3D models for these mRNAs, which exhibit similar tertiary structures. Binding of SELB to the fdhF mRNA hairpin was estimated to proceed with an apparent Kd of 30 nM. (+info)