The Heterogeneity of Bovine Pancreatic Ribonuclease S<sup>*</sup>

Doscher, Marilynn S.; Hirs, C.H.W.

doi:10.1021/bi00853a047

Cited by 90 publications

(35 citation statements)

References 19 publications

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“…It was noted previously (Doscher & Hirs, 1967) that S-protein is a mixture of RNase 21-124 and RNase 22-124 and that the use of this starting material for semisynthesis gives a mixture of RNase 1-124 and des21-RNase 1-124 (Homandberg & Laskowski, 1979). Therefore, we studied different subtilisins for their ability to cleave RNase A selectively at the Ala-20-Ser-21 bond.…”

Section: Subtilisin-catalyzed Semisynthesis Of Rnase a Variantsmentioning

confidence: 99%

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor

Neumann

Hofsteenge

1994

Protein Science

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Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisincatalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamidomethyl-cysteine, binding of recombinant ribonuclease inhibitor (RI) from porcine liver was strongly affected. In contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1 ,Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol". Modifications that introduced a positive charge in position 7 (S-aminoethyl-or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-' loss in binding energy. S-protein bound to R1 with K j = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol" difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.

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Section: Subtilisin-catalyzed Semisynthesis Of Rnase a Variantsmentioning

confidence: 99%

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor

Neumann

Hofsteenge

1994

Protein Science

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“…We previously (7,8) have prepared a semisynthetic ribonuclease-S' (SRNase-S')t, a noncovalent complex containing solid phase-derived synthetic fragment- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) the preparation of analogues (7,10,11). These studies have increasingly emphasized the desirability of being able to elucidate detailed structural features of semisynthetic analogues.…”

mentioning

confidence: 99%

“…RNase-S, the constituent fragments RNase-S-(1-20) and RNase-S-(21-124), and the reconstituted RNase-S' complex all were obtained from bovine pancreatic ribonuclease A (RNase-A) essentially as cited previously (7,12). The assay for RNase activity against cytidine 2':3'-cyclic monophosphate, as well as that for determination of amino acid composition of peptides after acid hydrolysis, have been described (7).…”

mentioning

confidence: 99%

“…The chemical synthesis of the peptide synthetic- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), corresponding to the normal sequence of the amino terminal RNase-S-(1-20) fragment, was carried out by the solid phase method (16,17), as modified previously for the synthesis of synthetic-(1-15) fragments (7). The protected Na-t-butyloxycarbonyl amino acid derivatives used, as well as the method for cleavage and deblocking, have been defined (7).…”

mentioning

confidence: 99%

“…Normal sequence SRNase-S' was prepared from crude synthetic- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and RNase-S-(21-124) by a procedure analogous to that developed for other SRNase-S' species (7,8). The synthetic and native polypeptides were added in a molar ratio of 1.3:1 and the resulting complex was purified by sulfoethylSephadex chromatography followed by desalting on Sephadex-G-25.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Crystalline semisynthetic ribonuclease-S'.

Pandin¹,

Padlan²,

DiBello³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Crystals of solid phase-derived semisynthetic ribonuclease-S' were prepared and compared with those for native ribonuclease-S' and -S. The semisynthetic species used was the noncovalent complex of synthetic fragment1-20), corresponding to residues 1 through 20 of bovine pancreatic ribonuclease-A (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22), and native ribonuclease-S-(21-124), the fragment containing residues 21 through 124 of ribonuclease-A. This semisynthetic complex was completely active enzymatically, was homogeneous as judged by polyacrylamide gel electrophoresis, and had no greater than trace amounts of excess ribonuclease-S-21-124) as judged by affinity chroma-

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Single- or Dual-Mode Switching of Semisynthetic Ribonuclease S′ with an Iminodiacetic Acid Moiety in Response to the Copper(II) Concentration

et al. 1999

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Abstract:Ribonuclease S' bearing iminodiacetic acid as a metal-binding site was designed and semisynthesized by self-assembly of native S-protein with chemically modified S-peptide. Iminodiacetic acid-appended amino acid (Ida 4 ) was synthesized and incorporated into the S-peptide sequence by solid-phase peptide synthesis based on Fmoc chemistry at a single site or double sites of the solvent-exposed side of the S-peptide. Circular dichroism (CD) spectroscopy of these S-peptides confirmed that the Cu II ion induced an increase or decrease of a-helix conformation depending on the replacement position. S-Peptide/Sprotein titration monitored by conventional enzymatic activity and UV or CD spectroscopy demonstrated that various S-peptides form a stable complex (Ida 4 ± RNase S') with S-protein, except when Met13 and Asp14 are replaced with Ida 4

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The Heterogeneity of Bovine Pancreatic Ribonuclease S^*

Cited by 90 publications

References 19 publications

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor

Crystalline semisynthetic ribonuclease-S'.

Single- or Dual-Mode Switching of Semisynthetic Ribonuclease S′ with an Iminodiacetic Acid Moiety in Response to the Copper(II) Concentration

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The Heterogeneity of Bovine Pancreatic Ribonuclease S*

Cited by 90 publications

References 19 publications

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor

Crystalline semisynthetic ribonuclease-S'.

Single- or Dual-Mode Switching of Semisynthetic Ribonuclease S′ with an Iminodiacetic Acid Moiety in Response to the Copper(II) Concentration

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The Heterogeneity of Bovine Pancreatic Ribonuclease S^*