Studies on extracellular ribonucleases of Ustilago sphaerogena. Purification and properties.
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ABSTRACT:
SUBMITTER: Arima T
PROVIDER: S-EPMC1198548 | biostudies-other | 1968 Feb
REPOSITORIES: biostudies-other
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Project description:1. Ribonuclease U(1) splits only the phosphodiester bonds of guanosine 3'-phosphates in RNA. It may be regarded as a guanyloribonuclease [ribonucleate (guanine nucleotide)-2'-transferase (cyclizing), EC 2.7.7.26] similar to ribonuclease T(1) (Egami, Takahashi & Uchida, 1964). It seems to be identical with the extracellular ribonuclease described by Glitz & Dekker (1963, 1964a,b). 2. Ribonucleases U(2) and U(3) are novel enzymes with a strict specificity. They split the internucleotide bonds between purine 3'-nucleotides and 5'-hydroxy groups of adjacent nucleotides in RNA with the intermediary formation of purine nucleoside 2',3'-(cyclic)-phosphates, which are slowly hydrolysed to purine 3'-nucleotides. So they may be classified as ;puryloribonucleases [ribonucleate (purine nucleotide)-2'-transferase (cyclizing)]'. Double-stranded RNA is scarcely split by ribonucleases U(2) and U(3). 3. Ribonuclease U(4) has no absolute base specificity, and produces the mononucleotides 3'-adenylate, 3'-guanylate, 3'-cytidylate and 3'-uridylate from RNA.
| S-EPMC1198549 | biostudies-other
Project description:The mammalian-type cytochrome c of the basidiomycete Ustilago sphaerogena contains in a single polypeptide chain of 107 residues, two histidine residues located at positions 18 and 33, and one methionine residue situated at position 80 (Bitar et al., 1972). The reaction of Ustilago ferricytochrome c with bromoacetate at neutral pH resulted in the modification of histidine-33, but not of histidine-18 or of the invariant methionine residue. The activities of Ustilago cytochrome c with mitochondrial cytochrome c oxidase and with NADH-cytochrome c reductase were unaltered by the modification. The equilibrium constants for the formation of low-spin complexes of the ferrihaem octapeptide of horse cytochrome c (residues 14-21, including the haem bound covalently to cysteines 14 and 17) with imidazole, N(2)-acetylhistidine and monocarboxymethyl derivatives of N(2)-acetylhistidine were determined spectrophotometrically. Alkylation of the imidazole side-chain group of N(2)-acetylhistidine resulted in a marked decrease in its ability to form low-spin ferrihaem complexes. These results indicate that in Ustilago ferricytochrome c in solution histidine-33 is not involved in the central co-ordination complex. Since side-chain groups of residues other than histidine and methionine do not appear to be involved in the central complexes of other mammalian-type cytochromes c (Hettinger & Harbury, 1964, 1965; Myer & Harbury, 1965) it is likely that in Ustilago ferricytochrome c in solution at neutral pH, the side-chain groups of histidine-18 and methionine-80 are involved in the central co-ordination complex. The latter is stable over the pH range 2.6-8.4.
| S-EPMC1166319 | biostudies-other
Project description:1. RNAase (ribonuclease) U2, a purine-specific RNAase, was reduced, aminoethylated and hydrolysed with trypsin, chymotrypsin and thermolysin. On the basis of the analyses of the resulting peptides, the complete amino acid sequence of RNAase U2 was determined, 2. When the sequence was compared with the amino acid sequence of RNAase T1 (EC 3.1.4.8), the following regions were found to be similar in the two enzymes; Tyr-Pro-His-Gln-Tyr (38-42) in RNAase U2 and Tyr-Pro-His-Lys-Tyr (38-42) in RNAase T1, Glu-Phe-Pro-Leu-Val (61-65) in RNAase U2 and Glu-Trp-Pro-Ile-Leu (58-62) in RNAase T1, Asp-Arg-Val-Ile-Tyr-Gln (83-88) in RNAase U2 and Asp-Arg-Val-Phe-Asn (76-81) in RNAase T1 and Val-Thr-His-Thr-Gly-Ala (98-103) in RNAase U2 and Ile-Thr-His-Thr-Gly-Ala (90-95) in RNAase T1. All of the amino acid residues, histidine-40, glutamate-58, arginine-77 and histidine-92, which were found to play a crucial role in the biological activity of RNAase T1, were included in the regions cited here. 3. Detailed evidence for the amino acid sequence of the sequence of the proteins has been deposited as Supplementary Publication SUP 50041 (33 PAGES) AT THE British Library (Lending Division)(formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.
| S-EPMC1165224 | biostudies-other
Project description:1. The complete amino acid sequence of cytochrome c from the basidiomycete Ustilago sphaerogena was determined from the amino acid compositions and sequences of either tryptic or chymotryptic peptides, and in homology with at least thirty other established sequences of cytochrome c. 2. The primary structure of the molecule bears all of the characteristics of a mammalian-type cytochrome c, showing the typical clustered distribution of hydrophobic and basic residues with a single polypeptide chain of 107 residues. 3. Like all other fungal cytochromes c, it possesses a free N-terminus, and one less residue at the C-terminus than vertebrate cytochromes c. The region of residues 70-80 is strictly conserved, as is histidine at position 18. Position 26 is occupied by an asparagine residue, in contrast to histidine which occurs at this location in most of the known sequences of mammalian-type cytochromes c. 4. In contrast to some other fungal and plant cytochromes c of known primary structures, the Ustilago cytochrome c molecule does not contain trimethyl-lysine. 5. The sequence of Ustilago cytochrome c differs from the sequences of human, horse, chicken, tuna, wheat, and baker's yeast proteins at loci 47, 43, 44, 44 and 38 respectively.
| S-EPMC1174159 | biostudies-other
Project description:Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.
| S-EPMC113159 | biostudies-literature
Project description:The apoplast of host plants serves as a dynamic interface during invasion and subsequent establishment of disease by a microbial pathogen. In the recent past, host plant apoplasts have been demonstrated to harbour small membrane bound vesicles called extracellular vesicles (EVs) that contain both RNA and protein. EVs are considered to play a significant role in cell-cell communication between the host and the pathogen during infection. In this study the RNAs associated with EVs from the host plant Zea mays under infected and uninfected conditions were sequenced. Besides EV bound RNA, plant apoplasts also contain free RNA that are equally important for host pathogen interaction. We have also detected a pool of extracellular RNA (exRNA) in Z. mays located on leaf surfaces. In this experiment, we sequenced both the free apoplastic RNA and leaf surface RNA from Z. mays during U. maydis infection and under control uninfected conditions. The U. maydis genome codes for a number of secreted ribonucleases that are essential for the pathogenesis of the fungus. For three of these ribonucleases, we have also investigated their role in the regulation of cell:cell communication between Z. mays and U. maydis. Accordingly, this study includes comparative transcript profiling of all the three different forms of exRNA between Z. mays plants infected with either wild type U. maydis or U. maydis strains lacking one or more secreted ribonucleases.
2024-09-17 | GSE277127 | GEO
Project description:Two RNAases from human cerebrum were purified to an electrophoretically homogeneous state and their molecular masses were 22.0 kDa (tentatively called RNAase HB-1) and 19.0 kDa (RNAase HB-2). Analyses of the amino acid compositions, N-terminal amino acid sequences and catalytic properties of these enzymes provided strong evidence that they were strictly related to the secretory (sec) RNAases, such as the pancreatic enzyme, very similar immunologically to urinary sec RNAase, but clearly distinguishable from urinary non-secretory (nonsec) RNAase. There were several differences between HB-1 and HB-2, namely their immunological reactivities with specific antibodies, heat-stabilities, attached carbohydrate moieties and molecular masses. In particular, HB-2 appeared to be nonglycosylated, in view of its lack of affinity for several conjugated lectins, the absence of hexosamine and no change in electrophoretic mobility before and after peptide:N-glycosidase F digestion, whereas HB-1 and human sec RNAases purified from kidney, pancreas and urine all appeared to be glycosylated, as they moved to the same position as HB-2 when electrophoresed after glycosidase digestion. An antibody against urinary sec RNAase inhibited 75% and 20% of the total activity of the crude cerebral extract against RNA at pH 8.0 and 6.0 respectively, whereas an antibody against urinary nonsec RNAase had no such inhibitory effect. These findings suggest that yet another type(s) of cerebral RNAase, which is unable to cross-react immunologically with sec and nonsec RNAases, may exist. Two RNAases corresponding to HB-1 and HB-2 were identified in fresh cerebrospinal fluid.
| S-EPMC1137742 | biostudies-other
Project description:Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.
| S-EPMC1164846 | biostudies-other
Project description:Extracellular vesicles (EVs) can transfer diverse RNA cargo for intercellular communication. EV-associated RNAs have been found in diverse fungi and were proposed to be relevant for pathogenesis in animal hosts. In plant-pathogen interactions, small RNAs are exchanged in a cross-kingdom RNAi warfare and EVs were considered to be a delivery mechanism. To extend the search for EV-associated molecules involved in plant-pathogen communication, we have characterised the repertoire of EV-associated mRNAs secreted by the maize smut pathogen, Ustilago maydis. For this initial survey, we examined EV-enriched fractions from axenic filamentous cultures that mimic infectious hyphae. EV-associated RNAs were resistant to degradation by RNases and the presence of intact mRNAs was evident. The set of mRNAs enriched inside EVs relative to the fungal cells are functionally distinct from those that are depleted from EVs. mRNAs encoding metabolic enzymes are particularly enriched. Intriguingly, mRNAs of some known effectors and other proteins linked to virulence were also found in EVs. Furthermore, several mRNAs enriched in EVs are also upregulated during infection, suggesting that EV-associated mRNAs may participate in plant-pathogen interactions.
| S-EPMC8306574 | biostudies-literature
Project description:Four ribonucleases (RNAases I-IV) have been purified to homogeneity from human seminal plasma by precipitation with 40-75%-satd. (NH4)2SO4, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose phosphocellulose, agarose-5'-(4-aminophenylphospho)uridine 2'(3')-phosphate (RNAase affinity column) and Sephadex G-75 or G-100. The homogeneity of these RNAases was confirmed by polyacrylamide-gel electrophoresis. Mr values for these purified RNAases were 78 000, 16 000, 13 300 and 5000 as estimated by gel filtration. Enzyme activities of RNAases I, III and IV were inhibited by Mn2+, Zn2+ and Cu2+ and activated by Na+, K+, Ba2+, Mg2+, Fe2+ and EDTA, whereas that of RNAase II was inhibited by Ba2+, Mg2+, Fe2+, Mn2+, Zn2+ and Cu2+ and activated by Na+, K+ and EDTA. RNAases I, II and IV demonstrated a higher affinity for poly(C) and poly(U) or yeast RNA, whereas RNAase III preferentially hydrolysed poly(U) over poly(C) and yeast RNA. In the presence of 5 mM-spermine, RNAase I was dissociated to a low-Mr (5000) enzyme with an increase in total RNAase enzymic activity. Xenoantiserum to each RNAase was raised and evaluated by immunoprecipitation and immunohistochemical methods. Anti-(seminal RNAase III) antiserum showed no immunological cross-reaction with RNAases of other human origin, whereas anti-(seminal RNAase I), -(RNAase II) and -(RNAase IV) antisera exhibited indistinguishable immunological reactions with serum RNAase and other human RNAases, except that anti-(seminal RNAase I) and -(RNAase antisera IV) did not react with pancreatic RNAases. Seminal RNAases I and IV were identical immunologically as shown by anti-(RNAase I) and anti-(RNAase IV) in immunodiffusion. Immunohistochemical study revealed that, among human tissues examined, only prostate expressed seminal RNAase III. These results suggested that human seminal RNAase I may be an aggregated molecule of RNAase IV and that seminal RNAases II and IV are similar to serum RNAases, whereas seminal RNAase III is a prostate-specific enzyme.
| S-EPMC1152442 | biostudies-other