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University of Groningen Horse pancreatic ribonuclease Scheffer, Albert Jan IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1973 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Scheffer, A. J. (1973). Horse pancreatic ribonuclease s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 19-06-2017 8. SUMMARY The.exocrine.pancreatic ribonucleases forn a group of relatively rapíd1y evolving proteins. Their disiribution anong the vertebrate species is exceptionally unequal; an acceptable explanation of this distribution by a Dossíb1e physiological function has been suggested ón1y'for aninals with ruminant or ruminant-1ike digestive systens. Many ribonucleases are glycosidated to a greater or lesser ext ent . Bovine ribonuclease is one of the most thoroughly studied enzymes. Comparative studies with ribonugleases derived from other species nay yield valuable information on their phylogeny and structure-function relationships, and on the specificity requirements for the types of oligosaccharide s ide-chain attached. Crude horse ribonuclease was isolated fron fresh tissue by acid extraction, phenol extraction a n d a r n n o n i u ms u l f a t e fractionation (4.1). Further purification of the enzyme was found to be difficult. After trying many modifications, we succeeded by using repeated chronatography on CM-cel1u1ose with three different gradient systens (4.2). The purity of the preparations was checked by disc ge1 elecand "staining" trophoresis for ribonucleolytic activity, and by N-terrninal analys is (4.3 , 4. 5); the specific activity was found to be an unreliable criterion for purity. Pure horse ribonuclease was both chromatographically and electrophoretically h e t e r o g e n e o u s . W e h a v e r n a d ep l a u s ible that the heterogeneity resides in the carbohydrate moiety (4. 8) . The amino acid composition of horse ribonuclease has the characteristics conmon to the other known pancreatic ribonucleases. Tryptophan is absent. The carbohydrate noiety contains glucosanine and hexoses (4.4). Determination of the molecular welght was difficult because of the carboby a conbination of methods we found a hydrate noietyi value of 14,000 for the nolecular weight of the protein noi.ety and about 18,000 for the average nolecular weight (4.7). Horse ribonuclease has the of the glycoprotein usual N-terninus lysine, but its C-terminus is threonine instead of the usual valine. With spectrophotometric tiwe found only two abnormal- tyrosines tration and three normal ones (4.6). tto We derived four qeries nf nontids5 from horse ribonuclease (chapter 5) : rc (;;i;;i;^"í;";hynotryptic digesti.on after perfornic acid oxidation), T Itryptic cleavage after perforrnic acid oxidation), CN (cyanogen bronide cleavage followed by perfornic acid oxidation), and AET (tryptié iiydrolysis after reduction and aminoethvlation). Sóme secondary cleavages were perforned with thermolysin, chyrnotryptil: and cyanogen bromide. Peptides were isolatecl and pu: rified by ge1 filtration, chrornatography on various ion exchangers, and column electrophoresis. The latter methocl proved particularly useful for glycopeptides (3.5.5). For the detection of-peptides in colrrmn eluates we used mainly spectrophotornetric rnonitoring, manual ninhydrin analys is ,and automated analysis with a considerably modified peptide analyser.that has been described at some length (3. 5.7). The purity of the peptides was checked by high vo1tage paper electrophoresis, amino acid analysis and dansylation. Peptide sequences were determined-by conventional nethods: two versions of the Dansyl-Edrnan technique and a nrodification o f t h e d i r e c t E d n a n d e s r a d a t i o n f 3 . ó ') . Only the AET-peptides covered the entiie s"q.l"n.è. Several of the CN-peptides had arisen frorn aspecific cleavages, especially to the right of tyrosine residues. All peptides were positioned by sequence homology and overlappings; all but four peptide bonds were overlapped by one or nore peptides (6.2). Alnost al1 amide groups were l o c a t e d u n e q u i v o c a l l y ( 6 . 1) . Horse ribonuclease consists of a polypeptide chain containing 125 anino acid re idues and carrying three oligosaccharide side-chains in positions 21, 34, and 62. part of Asn-2.1 occurs in the carbohydrate-free forn (6.4). All three sites conforn to the postulated recognition triplet for glycosidation; site 21-23 is the first attachment site in any glycoprotein found to contain proline (7.3). The C-terminus of horse ribonuclease contains two additional arnino acids, representing the only instance of a C-terminal addition in pancreatic ribonucleases observed thus far. Another peculiarity unprecedented in pancreatic ribonucleases is the deletion of residue 39. Including deletion and addition, horse ribonuc lease differs in 35 positions fron the bovine enzyme (7 .1). All substitutions, the addition, and the oligosaccharide residues can be acconmodated in the three-dinensional nodel of bovine ribonuclease-S without alterating the folding of the backbone. The nodel built of the loop containing the deletion offered an elegant explanation for the deviating titration behaviour of horse ribonuclease: the shortening and consequent distortion of this loop prevents Tyr-92 fron forming a hydrogen bridge between its phenolic hydroxyl group and the carbonyl oxygen of residue 37 (7.2). 127 Comparison of all ribonuclease sequences elucidated partly to conthus far provided the infornation or èonpletely tree and a hypothetical evolutionary a preliminary struct riboamino acid sequence of narnmalian pancreatic ancestral nuclease (7 .1) . ),n1\à \6 b Qn A- @ Fig. B. Ancestral 128 mammal