Purification by Affmity Chromatography and Physicochemical Properties of the Guanine-Specific Ribonuclease of Fusarium moniliforme1

Abstract

Ribonuclease F1, the guanine-specific ribonuclease of Fusarium moniliforme, was purified to homogeneity by a combination of ethanol fractionation, affinity chromatography and DEAE-cellulose column chromatography. The adsorbent for the affinity chromatography was synthesized by the coupling of periodate-oxidized guanosine 5'-monophosphate to aminohexyl agarose followed by sodium borohydride reduction. Ribonuclease F2, the minor component, was also purified to near homogeneity by the same procedure. Ribonucleases F1 and F2 had the same molecular weight (about 11,000) as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They also showed the same amino acid composition and differed only in the isoelectric point: 4.10 for F1 and 3.96 for F2.

Characterization and comparison of a Neurospora crassa RNase purified from cultures undergoing each of three different states of derepression

“…The properties of N4 resemble those of the enzymes listed, although the molecular weight of N4 is lower. The others have molecular weights ranging from 11,000 to 11,500 (7,25). N1 was reported as a stationary-phase enzyme (10, 20) and has similar properties to those reported here for N4 except for differences in size and amino acid composition (7,22).…”

“…Purer preparations and further characterization will be necessary to determine specificity and whether the enzymes are identical in every respect. Multiple formns of the same RNase have recently been reported for three different fungal organisms (11,23,25). In these cases, amino acid composition was the same, but differences in isoelectric points, conformations, or specific activities were observed.…”

“…Assuming 95% purity, most values approximate the correct number of residues. The composition of N4 was very similar to that of N1 and other guaninespecific RNases (25). N4 had fewer residues than N1, where differences are apparent, commensurate with its smaller size: N1 = 11,450 (7).…”

Comparison of the primary structures of ribonuclease U2 isoforms

“…However, this was not a valid possibility for the enzyme RNAase U2, which is nonglycosylated. The presence of isoforms was found in other guanine-specific RNAases, namely RNAase T1 from Aspergillus oryzae [5] and RNAase F1 from Fusarium moniliforme [6]. In each case the isoform enzyme has not been isolated from the original one and its enzymic and physicochemical properties are still unclear.…”

Microbial ribonucleases (RNases): production and application potential

“…Reducing glucose, peptone and soybean concentrations of the medium stimulated the RNase production from immobilized cells by 2.3 and 2.5 times higher than that of free cells in a bioreactor and flasks, respectively (Manolov 1992b). Molecular structure and characterization of RNases from different fungal producers were also investigated including guanyl-specific RNase from Penicillium chrysogenum (Yakovlev et al 1980) and Penicillium brevicompactum (Shlyapnikov et al 1984), guanyl-specific RNase FI1 from Fusarium moniliforme (Yoshida et al 1980), base-specific RNase Lu from A. niger (Horitsu et al 1982), RNase C 2 from A. clavatus (Bezborodova et al 1983), minor RNase from Aspergillus saito (Flögel et al 1988), RNase T1 from A. oryzae (Koepke et al 1989), RNase Rh from Rhizopus niveus (Kurihara et al 1992), RNase Th from Trichoderma harzianum (Vasileva-Tonkova 1993; Vasileva-Tonkova and Bezborodova 1996), novel poly(U) and poly(C) RNase from Saccharomyces cerevisiae (Lalioti et al 1997), atypical (guanylic acid preferential) extracellular RNase T2 from R. stolonifer (Chacko and Shankar 1998), a-sarcin from A. giganteus (Perez-Canadillas et al 2000), new type RNase T2 from Lentinus edodes and Irpex lacteus (Kobayashi et al 2003), extracellular poly(A) specific RNase from A. niger (Gundampati et al 2011a), recombinant RNase U2 from U. sphaerogena (Alvarez-Garcia et al 2009) and recombinant mitogillin from Aspergillus fumigatus (Kao et al 1998). …”