RIBONUCLEASES FROM THE EXTREME THERMOPHILIC ARCHAEBACTERIUM S-SOLFATARICUS

被引:43
作者
FUSI, P
TEDESCHI, G
ALIVERTI, A
RONCHI, S
TORTORA, P
GUERRITORE, A
机构
[1] UNIV MILAN,DIPARTIMENTO FISIOL & BIOCHIM GEN,VIA CELORIA 26,I-20133 MILAN,ITALY
[2] UNIV MILAN,IST FISIOL VET & BIOCHIM,I-20133 MILAN,ITALY
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1993年 / 211卷 / 1-2期
关键词
D O I
10.1111/j.1432-1033.1993.tb19899.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A purification procedure consisting of DEAE-Sephacel chromatography, heparin-Sepharose CL6B 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 pl, 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.
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页码:305 / 310
页数:6
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