Purification, some properties, and primary structure of base non-specific ribonucleases from Physarum polycephalum.
Two ribonucleases (RNase Phya and RNase Phyb) were purified to homogeneity on SDS-PAGE from the culture filtrate of the fungus Physarum polycephalum. The apparent molecular weights of RNases Phya and Phyb were about 20,000. The pH optima of these two RNases were around 4.5-4.75. The RNases released mononucleotides from RNA in the order of 3'-GMP, 3'-AMP, and 3'-pyrimidine nucleotides. RNase Phya and RNase Phyb have the N-terminal amino acid sequences STSFD--- and KSTSF--, respectively. This finding and the similar amino acid compositions of both RNases indicated that they might share the same protein moiety except for the N-terminus Lys. The complete primary structure of RNase Phyb was determined, mostly by analysis of the peptides generated by trypsin, V8 protease, and lysylendopeptidase digestions. The molecular weight of the protein moiety was 19,704. The locations of four half cystine residues were almost superimposable on those in five known fungal RNase T2 family RNases, but two others were not. The sequence homology between RNase Phyb and five known fungal RNases amounted to 53-59 residues, which are concentrated around the three histidine residues, supposed to form the active site in enzymes of the RNase T2 family. However, the amino acid sequence of RNase Phyb more closely resembles those of plant RNases such as RNases from Nicotiana alata [McClure, B.A. et al. (1989) Nature 342, 955-957], tomato [RNase Le, Yost et al. (1991) Eur. J. Biochem. 198, 1-6], and Momoridica charantia [RNase MC1, Ide et al. (1991) FEBS Lett. 284, 161-164].
Department of Microbiology, College of Pharmacy, Nihon University, Chiba.
N
N
Inokuchi
T
T
Koyama
F
F
Sawada
M
M
Irie
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016839:Physarum polycephalum / Q000201:enzymology*; D011506:Proteins / Q000032:analysis; D012260:Ribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D011506:Proteins; D012260:Ribonucleases