Aphanasenko G.

Dudkin S.

Kaminir L.

Leshchinskaya I.

Severin E.

FEBS Letters

Kazan Federal University Digital Repository

Volume 97, number 1 FEBS LETTERS January 1979 PRIMARY STRUCTURE OF RIBONUCLEASE FROM BACILLUS INTERMEDIUS 7P G. A. APHANASENKO, S. M. DUDKIN, L. B. KAMINIR, I. B. LESHCHINSKAYA*, E. S. SEVERIN Instuute of Molecular Biology, USSR Academy of Sciences, Moscow, II 7984 and *Ka.zan State University, Kazan, 420008, USSR Received 10 October 1978 1. Introduction Stmhes on the relation between the structure and function of RNases have been carried out mainly with RNase A [ 1 ] and to a lesser extent wrth RNase Tr [2] . Bacterial RNases have been significantly less stu&ed m this respect. Up to now, the primary structure and some physrcochemical properties of only one bacterial RNase, namely the RNase of B. amyloliquefaciens, have been established [3-S]. The purpose of this study was to determine the primary structure of the extracellular RNase of Bacillus intennedius, strain 7P. The RNase catalyses the breakdown of RNA to oligonucleotides ter- mmating in 3’-phosphate and 1s mainly specific towards the purine bases of the RNA molecule [6] . Nucleoside3’*3’-phosphates nddinucleoside phosphates are completely renstant o the action of thrs enzyme. The large scale preparation of the homogeneous RNase and some characteristics of this enzyme were described m [7] . 2. Materials and methods The ammo acid composition of the protein and peptrdes was determined using a Bra-Cal BC-201 ammo acid analyser (GFR). The ammo acid sequence of the N-terminal part of the protein was established on a Beckman 890C automatic sequenator. PTH derivatives of the amino acids were determined by thin-layer chromatography on polyamide plates [8] with subsequent scannmg on a PMQ-II spectrophotom- eter (Opton, GFR) and processing of results according to [9] , by gas-liquid chromatography [lo] , ElsevlerjNorth-Holland Blomedxal Press and by ammo acid analysis after hydrolysis of PTH derivatives of amino acids with 57% HI [ 1 l] . The sequence of peptides was determined by the dansyl-Edman technique and using carboxypeptidases A, B and C and leucine ammopeptidase. The kinetics of the exopeptidase degrad$tron of peptides were studied by means of the quantitative method of amino acid analysis based on their dansylation, followed by thin-layer chromatography on poly- amide plates [ 121. DNS-amino acids were scanned using an automatic system \yith a Saratov mini- computer [131 .Hydrolysis of the protein with the protease from the V8 stram of Staphylocwcus aureus was per- formed in 0.1 M NbHCOs (pH 8.0) at 37OC for 15 h [ 141 at an enzyme-substrate ratio 1.30. Cleavage of the RNase tryptophanyl bonds was carried out with N-chlorosuccimmrde according to [ 151. 3. Results and discussion RNase of B. mtermedius, stram 7P, has mol. wt -12 300 and contains 109 ammo acid residues [7]. The native protein was etiined by automatic phenyl isothrocyanate degradation, and 40 out of the first 50 residues were unequivocally identified. The remammg residues were rdentlfled upon analysrs of the peptides. The mrxture of peptrdes obtamed after hydrolysis of RNase with Staphylococcus aureus protease was fractionated by gel filtration on a Sephadex G-50 (fine) column m 10% acetic acid, contammg 8M urea. The resulting fractions were further purified by ion-exchange chromatography on an SP-Sephadex 77 

Primary structure of ribonuclease from bacillus intermedius 7P

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Primary structure of ribonuclease from bacillus intermedius 7P

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