Ribonucleases from the extreme thermophilic archaebacterium S. solfataricus.
A purification procedure consisting of DEAE-Sephacel chromatography, heparin-Sepharose CL-6B chromatography and Mono-S chromatography led to the isolation of three proteins endowed with RNase activity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. They were referred to as p1, p2 and p3, according to their elution order from the Mono-S column. Complete amino acid sequence of p2 and partial sequence of p3 displayed high sequence similarity to the 7-kDa DNA-binding proteins previously isolated in Sulfolobus strains [Choli, T., Wittman-Liebold, B. & Reinhardt, R. (1988) J. Biol. Chem. 263, 7087-7093]. The molecular mass of p2, calculated from sequence data, was 7.02 kDa, which compares fairly well with the value of 7.4 kDa determined by SDS/PAGE. Gel filtration of the molecule under native conditions displayed, however, a largely prevailing form with an assessed molecular mass of 13.0 kDa, which points to a dimeric structure. Kinetic characterization of protein p2 showed a broad pH optimum in the range 6.7-7.6 using yeast RNA as substrate; also, it was shown that activity was unaffected by EDTA, Mg2+ and phosphate. The enzyme did not accept as substrate any homopolyribonucleotide, which points to a rather narrow substrate specificity. This was also confirmed by incubating p2 with tRNA(fMet)Met (fMet, N-formylmethionine) from Escherichia coli: the hydrolysis products were thus identified as 3'-phosphooligonucleotides.
Fusi
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P
Dipartimento di Fisiologia e Biochimica generali, Università di Milano, Italy.
Tedeschi
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G
Aliverti
A
A
Ronchi
S
S
Tortora
P
P
Guerritore
A
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D016944:Sulfolobus / Q000201:enzymology*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012260:Ribonucleases