Studies on extracellular ribonucleases of Ustilago sphaerogena. Purification and properties

1. Four ribonucleases were isolated from culture media of Ustilago sphaerogena. They were designated ribonucleases U(1), U(2), U(3) and U(4). 2. They were purified about 1600-, 3700-, 1100- and 16-fold respectively. 3. It was shown by gel filtration that ribonucleases U(1), U(2) and U(3) have molecular weights about 10000 like ribonuclease T(1), and that ribonuclease U(4) is much larger. 4. Ribonucleases U(1), U(2) and U(3) are thermostable, but ribonuclease U(4) is not. 5. The pH optimum of ribonucleases U(1) and U(4) is pH8.0-8.5, and that of ribonucleases U(2) and U(3) is pH4.5.     

The disulfide bridges of ribonuclease U2 from Ustilago sphaerogena.

RNase U2 was partially hydrolyzed with chymotrypsin [EC 3.4.21.1] and sulfuric acid, and in each case the resulting peptides were separated by gel filtration, ion exchange column chromatography and paper electrophoresis. From the results of amino acid analysis of cystine-containing peptides and their oxidized components, the three disulfide bridges were located between the cystine residues at positions 1 and 53, 9 and 112, and 54 and 95.     

Ethoxyformylation of ribonuclease U2 form Ustilago sphaerogena.

RNase U2 was inactivated by incubation with ethoxyformic anhydride at pH 6.0 and pH 4.5. The absorbance of the RNase U2 increased at around 250 nm and decreased at around 280 nm. The inactivation occurred in parallel with the amount of modified histidine and plots of the relationship between the remaining activity and the modified histidine suggested that the modification of one of the two histidine residues totally inactivated the enzyme. The inactivated enzyme RNase U2 was reactivated by a low concentration of hydroxyamine, with removal of the ethoxyformyl group from the modified histidine residue. At pH 4.5, 2'-adenylate and 2'-guanylate protected RNase U2 from inactivation by ethoxyformic anhydride. The difference CD spectra showed that the ability of RNase U2 to form a complex with 2'-adenylate was lost on ethoxyformylation.     

The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena.

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.     

Ribonuclease inhibitor from human placenta. Purification and properties

A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion exchange and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. In common with the inhibitors of pancreatic ribonuclease from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:1 complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 X 10(-10) M. The amino acid composition of the protein has been determined. The protein contains 30 half-cystine plus cysteine residues determined as cysteic acid after performic acid oxidation. At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dissociation of the RNase - inhibitor complex into active RNase and inactive inhibitor.     

Iron uptake from ferrichrome A and iron citrate in Ustilago sphaerogena.

Double radioactive label transport assays with iron, chromium, and gallium chelates were used to investigate the mechanism of iron uptake by Ustilago sphaerogena. In iron-deficient cells, ferrichrome A iron was taken up without appreciable uptake of the ligand. Iron-sufficient cells partially accumulated the ligand with the metal. The chromium- and gallium-containing analogs of ferrichrome A were transported as intact chelates. Ferrichrome A iron uptake was inhibited by dipyridyl. The data suggest that the intact ferrichrome A chelate binds to a specific receptor, the iron is then separated from the ligand at the membrane by reduction, and the metal is released to the inside of the cell while the ligand is released to the exterior. The reduction step is not transport rate limiting. Iron chelated to citrate was taken up by an energy-dependent process. The citrate ligand was not taken up with the metal. Uptake was sensitive to dipyridyl and ferrozine. Chromic ion chelated to citrate was not transported, suggesting that the iron, rather than the chelate, is recognized by the receptor or that reduction of the metal is required for transport.     

The amino acid sequence of ribonuclease U1, a guanine-specific ribonuclease from the fungus Ustilago sphaerogena

The complete amino acid sequence of ribonuclease U1 (RNase U1), a guanine-specific ribonuclease from a fungus, Ustilago sphaerogena, was determined by conventional protein sequencing, using peptide fragments obtained by several enzymatic cleavages of the performic acid-oxidized protein. The oxidized protein was first cleaved by trypsin and the resulting peptides were purified and their amino acid sequences were determined. These tryptic peptides were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of the oxidized protein. The amino acid sequence thus deduced was further confirmed by isolation and analysis of peptides obtained by digestion of the oxidized protein with lysyl endopeptidase. The location of the disulfide bonds was deduced by isolation and analysis of cystine-containing peptides from a chymotryptic digest of heat-denatured RNase U1. These results showed that the protein is composed of a single polypeptide chain of 105 amino acid residues cross-linked by two disulfide bonds, having a molecular weight of 11,235, and that the NH2-terminus is blocked by a pyroglutamate residue. It has an overall homology with other guanine-specific or related ribonucleases, and shows 48% identity with RNase T1 and 38% identity with RNase U2.     

The primary structure of cytochrome c from the rust fungus Ustilago sphaerogena

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.     

The role of ferrichrome reductase in iron metabolism of Ustilago sphaerogena.

Ferrichrome, the ferric ionophore for Ustilago sphaerogena, can serve as a source of iron for the enzyme ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) in this organism, but only after enzymatic removal of the iron from its carrier. U. sphaerogena contains a specific ferrichrome reductase (NADH:ferrichrome oxidoreductase) which catalyzes cellular dissociation of the complex by reduction of the metal to the ferrous state. A spectrophotometric assay was developed based on trapping of the ferrous ion produced by ferrozine. There is an apparent inhibition by oxygen which is thought to be due to re-oxidation of the metal under the assay conditions. The close structural analogue, ferrichrome A, is not a substrate, nor is the ester type siderochrome ferric hexahydro-N,N',N"-triacetylfusarinine C. Aluminum desferriferrichrome is inhibitory. The importance of this enzyme for the metabolism of iron in this organism is discussed.     

Role of two siderophores in Ustilago sphaerogena. Regulation of biosynthesis and uptake mechanisms

Under iron-deficient conditions the smut fungus Ustilago sphaerogena produces two kinds of siderophores, ferrichrome and ferrichrome A. Regulation of ligand biosyntheses and uptake mechanisms of the iron chelates were studied to determine the role of each chelate in U. sphaerogena. The biosynthesis of each ligand was differentially regulated. Ferrichrome A, the more effective chelate, was preferentially synthesized under more extreme conditions of iron stress, but completely repressed when the cell was supplied with sufficient iron. In contrast, biosynthesis of ferrichrome was strongly but not completely repressed by iron. The mechanism of repression was examined using a newly developed in vivo synthesis assay. Chromium and gallium-containing siderophore analogs had no effect on siderophore ligand biosynthesis. Iron, added as siderophores, resulted in increased oxygen uptake and amino acid transport, which was soon followed by decreased ligand biosynthesis, suggesting that regulation may be indirect and related to oxidative metabolism. Uptake experiments were used to rule out a ligand-exchange mechanism for ferrichrome A-iron transport. The data suggest that ferrichrome A-iron is taken up at a specific site that results in a rapid distribution of iron inside the cell.     

Purification and properties of deoxyadenosine kinase from rat liver mitochondria. I. Purification and physical properties

Deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76, AdR kinase) from rat liver mitochondria has been partially purified and compared with partially purified AdR kinase from the cytosol of the same biological material. Some physical properties of both enzymes, including molecular weight, gel electrophoresis and gel isoelectric focusing were investigated and considerable differences between these data for mitochondrial and cytosol AdR kinase were found.     

A DNA polymerase from Ustilago maydis. 1. Purification and properties of the polymerase activity.

A DNA polymerase from Ustilago maydis has been purified to apparent homogeneity. The native enzyme possesses a subunit structure consisting of 50000 and 55000-dalton monomers. The apparent sedimentation coefficient of the polymerase activity in the absence of salt is 8.4 S (Mr=180000-200000), that in its presence (0.6 M NaCl or 0.12 M KCl) being 6.3 S (Mr=80000-100000). Low concentrations of EDTA also converted the 8.4-S to a 6.3-S form, whereas magnesium ions catalysed the reverse association. The enzyme has an absolute requirement for both a DNA or RNA template and a DNA primer. For homopolymer templates the primer requirement was satisified by a short complementary oligodeoxynucleotide, but oligoribonucleotides were extremely inefficient primers. With the template-primer poly(dA) X (dT)12, the enzyme added an average of 50 dTMP nucleotides on to each primer molecule, whereas with poly(rA) X (dT)12, this figure was 300. The enzyme also possesses an associated deoxyribonuclease activity. No other DNA polymerase activity was detected in cell-free extracts of U. maydis.     

Acetate kinase from Veillonella alcalescens. Purification and physical properties.

Acetate kinase (ATP:phosphotransferase E.C.2.7.2.1) has been purified to a high state of purity from Veillonella alcalescens. The native enzyme had a molecular weight of 88,000, as determined by Sephadex G-150 gel filtration. The molecular weight of the monomeric enzyme, estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42,000. The enzyme was determined to be a homodimer from the amino acid composition and the results of trypsin digestion and cyanogen bromide cleavage. Two moles of phosphate were incorporated into the dimer upon incubation of the enzyme with ATP and acetate. These results support the conclusion that each subunit of the dimeric enzyme consists of a single active catalytic center. Succinate enhanced the rate of ATP-ADP phosphoryl group exchange 20-fold and the binding of ATP 10-fold. These results are considered in light of data from previous reports (Pelroy, R. A., and Whiteley, H. R. (1971) J. Bacteriol. 105, 259-267; Bowman, C. M., Valdez, R. O., and Nishimura, J. S. (1976) J. Biol. Chem 251, 3117-3121).     

Nonspecific acid phosphatase from Schizosaccharomyces pombe. Purification and physical chemical properties.

Repressible nonspecific acid phosphatase from Schizosaccharomyces pombe was purified to apparent homogeneity, as ascertained from ultracentrifugal, electrophoretic, and chromatographic data. The native protein has a molecular weight of 383,000 as determined by sucrose density gradient centrifugation and 381,000 as determined by gel filtration. The native protein can be dissociated in the presence of 8 M urea-1% sodium dodecyl sulfate into sub-units possessing an approximate molecular weight of 104,000. Neutral sugars account for about 66% of the total molecular weight and contribute to the high solubility and some of the other physical properties of this enzyme. Purified enzyme preparations have a Km for 4-nitrophenyl phosphate of 0.17 mM and a broad substrate specificity, but do not show diesterase activity. Phosphate and sulfate are competitive inhibitors. The enzyme is inactivated at neutral and alkaline pH and at relatively low temperatures. Mannose and galactose was found as the main components of the carbohydrate moiety; glucosamine was present in lower amounts. The amino acid analysis revealed a high content of aspartate, threonine, and serine; no sulfhydryl group could be detected. Pi is released in stoichiometric amount (1 mol per enzyme monomer) on protein digestion.     

Role of two siderophores in Ustilago sphaerogena: Regulation of biosynthesis and uptake mechanisms

Under iron-deficient conditions the smut fungus Ustilago sphaerogena produces two kinds of siderophores, ferrichrome and ferrichrome A. Regulation of ligand biosyntheses and uptake mechanisms of the iron chelates were studied to determine the role of each chelate in U. sphaerogena. The biosynthesis of each ligand was differentially regulated. Ferrichrome A, the more effective chelate, was preferentially synthesized under more extreme conditions of iron stress, but completely repressed when the cell was supplied with sufficient iron. In contrast, biosynthesis of ferrichrome was strongly but not completely repressed by iron. The mechanism of repression was examined using a newly developed in vivo synthesis assay. Chromium and gallium-containing siderophore analogs had no effect on siderophore ligand biosynthesis. Iron, added as siderophores, resulted in increased oxygen uptake and amino acid transport, which was soon followed by decreased ligand biosynthesis, suggesting that regulation may be indirect and related to oxidative metabolism. Uptake experiments were used to rule out a ligand-exchange mechanism for ferrichrome A-iron transport. The data suggest that ferrichrome A-iron is taken up at a specific site that results in a rapid distribution of iron inside the cell.