Studies with the S-peptide-S-protein system: The role of glutamic acid-2, lysine-7, and methionine-13 in S-peptide1–14 for binding to and activation of S-protein

Hofmann, Klaus; Andreatta, R. H.; Finn, Frances M.; Montibeller, Judith; Porcelli, G; Quattrone, Alfred J.

doi:10.1016/0045-2068(71)90007-1

Cited by 22 publications

(10 citation statements)

References 34 publications

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“…The combination of RNase(l-l 18) (140 nM) and [Ala114]Ang(108-123) (80 ^M) also shows no activity toward CpA. Competition assays (Hofmann et al, 1971) using CpA as substrate reveal that the binding of [Ala13]Ang(l-21) to the S-protein is about 50-fold weaker than that of Ang(l-21) while [Ala114]Ang(108-123) binds to RNase(l-118) about 1.5-fold more weakly than the parent peptide (data not shown). These results indicate that complex formation would have been at least 50% under the assay conditions employed.…”

Section: Resultsmentioning

confidence: 99%

Enzymically active angiogenin/ribonuclease A hybrids formed by peptide interchange

Jw¹,

Ds²,

Jf³

et al. 1988

Biochemistry

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The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

Enzymically active angiogenin/ribonuclease A hybrids formed by peptide interchange

Jw¹,

Ds²,

Jf³

et al. 1988

Biochemistry

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“…Lys-7 and Lys-66 have also been observed to be positioned in the active site region. While both of these residues are absent in angiogenin, it is known that neither is essential for RNase activity (Hofmann et al, 1971;Beintema et al, 1985).…”

Section: Discussionmentioning

confidence: 99%

Characteristic ribonucleolytic activity of human angiogenin

Shapiro¹,

Riordan²,

Vallée³

1986

Biochemistry

346

314

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Angiogenin, a blood vessel inducing protein isolated from a human tumor cell line, has been found to exhibit ribonucleolytic activity. It catalyzes the cleavage of both 28S and 18S ribosomal RNA as determined by agarose gel electrophoresis. The major products formed with these substrates are 100-500 nucleotides in length. In contrast, angiogenin is inactive toward all of the more conventional substrates of the homologous pancreatic ribonucleases. In particular, it does not produce detectable amounts of acid-soluble fragments from high molecular weight wheat germ RNA, poly(C), or poly(U), nor does it hydrolyze cytidine or uridine cyclic 2',3'-phosphate. The high degree of sequence homology between angiogenin and the pancreatic ribonucleases, which includes all three catalytic residues, His-12, Lys-41, and His-119, has thus identified the chemical nature of a potential angiogenin substrate. These results may bear importantly on the physiological function of angiogenin.

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“…Dissociation constants were in the range of 10-4-10-5 m. Structural modifications of S-peptide which have a marked influence on S-protein activation or RNase S inhibition in the presence of substrate are T A he discovery of the S-peptide-S-protein system* 1 (Richards, 1958) provided a unique opportunity to measure a number of parameters of peptide-protein interactions. The fortuitous location of part of the active site in the S-peptide portion of ribonuclease S permitted synthetic manipulations to be made leading eventually to the discovery of a number of potent inhibitors (Finn and Hofmann, 1967;Hofmann et al, 1970Hofmann et al, ,1971. Furthermore a systematic replacement of residues along the peptide chain has allowed definition of amino acids contributing to the strong, highly specific, noncovalent binding between peptide and protein.…”

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confidence: 99%

“…The generation of a difference spectrum upon addition of S-peptide to S-protein was first noted by Richards and H-Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Met-Asp-Ser-Ser-Thr-Ser-Ala-Ala-OH Dissocn Constant, » The 50% ratio refers to enzyme activity measurements using RNA as substrate (Finn and Hofmann, 1965).» Mole ratio of peptide to inhibitor (Pyr12S-peptidei_l4) required to regenerate 50% of the activity of RNase S. = Moles of peptide required to inhibit 50% the activity of 1 mole of RNase S. d Mole ratio of peptide to inhibitor (Pyr12S-peptideW4) required to regenerate 50% of the activity of the complex Nle13S-peptide,_,4-S-protem; this complex is not as active as RNase S (see Hofmann et al, 1971). Ultraviolet difference spectra for the reassociation of Sprotein and synthetic S-peptide and three analogs.…”

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confidence: 99%