Wim Gaastra

Affinity chromatography of porcine pancreatic ribonuclease reinvestigation of the N-terminal amino acid sequence.

In 1969 Wilchek et al. [I] described the affinity chromatography of bovine ribonuclease A using Agarose to which a strong competitive inhibitor of ribonuclease, 5’-(4-aminophenyl)-uridine-(2’,3’) phosphate (APUP) was coupled. On this adsorbent binding occurred at low ionic strength and neutral pH; desorption was achieved with 2 M acetic acid. We were unable to repeat their results, but could obtain desorption of ribonuclease with 4 M NaCl. Also, with crude pancreatic extracts, we could only achieve satisfactory binding of the enzyme at an ionic strength of 0.2, which largely prevents aspecific interactions. In this way we have already isolated pure ribonucleases from more than fifteen mammalian species in sufficient amounts for amino acid sequence studies. In this article we describe the purification of porcine ribonuclease. Jackson and Hirs [2] have determined the amino acid sequence of this enzyme. In the N-terminal sequence they found glutamine at the positions 2 and 9, where, in other pancreatic ribonucleases, normally glutamic acid is found, (except for rat [3] which contains lysine at position 9). The side chain of glutamic acid 9 has no distinct function in the structure of bovine ribonuclease, but glutamic acid 2 may form a salt brdige with the side chain of arginine 10 [4], both “constant” residues in pancreatic ribonucleases. Marchiori et al. [5] and Hofmann et al. [4, 61 have demonstrated in their synthetic S-peptide studies that if this ion pair cannot be formed, the resulting partially synthetic ribonucleases S’ are less active. Therefore, we reinvestigated the state of amidation of glutamic acid residues in the N-terminal part of porcine ribonuclease.

The molecular evolution of pancreatic ribonuclease.

SummaryThe primary structures of pancreatic ribonucleases from 26 species (18 artiodactyls, horse, whale, 5 rodents and turtle) are known. Several species contain identical ribonucleases (cow/bison; sheep/goat), other species show polymorphism (arabian camel) or the presence of two structural gene loci (guinea pig pancreas contains two ribonucleases that differ at 31 positions). 26 different sequences (including the ribonuclease from bovine seminal plasma which is paralogous to the pancreatic ribonucleases) were used to construct a most parsimonious tree. A second tree that most closely approximates current biological opinion requires 402 whereas the most parsimonious tree requires 389 nucleotide substitutions. The “artiodactyl” part of the most parsimonious tree conforms quite well with the biological one of this order, except for the position of the giraffe which is placed with the pronghorn. Other parts of the most parsimonious tree agree less with the biological tree, probably as a result of the occurrence of many parallel and back substitutions. Bovine seminal ribonuclease was found to be the result of a gene duplication which occurred before the divergence of the true ruminants, but after the divergence of this group from the cameloids.The evolutionary rate of ribonuclease was found to be 390, 3.0 and 11 nucleotide substitutions per 109 yrs per ribonuclease gene, codon and covarion respectively. However, there is much variation in evolutionary rate in different taxa. Values ranging from about 100 (in the bovidae) to about 700 (in the rodents) nucleotide substitutions per 109 yrs per gene were found.A method for counting parallel and back mutations is presented. The 389 nucleotide substitutions in the most parsimonious tree occur at 88 codon positions; 154 of them are the result of parallel and back mutations. Parallel evolution to a similar structure, including the presence of 2 sites with carbohydrate, was demonstrated in an extensive region at the surface of pig and guinea pig ribonuclease B. The presence of carbohydrate probably is important in a number of species. A correlation between the presence of heavily glycosidated ribonucleases and coecal digestion was observed. Hypothetical sequences of ancestral ungulate ribonucleases contain many recognition sites for carbohydrate attachment; this suggests that herbivores with coecal digestion might have preceded the true ruminants in mammalian evolution.

The primary structure of giraffe pancreatic ribonuclease

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Partial covalent structure of two basic chromosomal proteins from human spermatozoa.

The partial covalent structure of the basic chromosomal proteins I and II isolated from human spermatozoa was determined by automatic Edman degradation and digestion with carboxypeptidases A and B. The partial covalent structures obtained are compared with complete and partial known sequences of the basic chromosomal proteins from other animals.

The preparation and primary structure of S-peptides from different pancreatic ribonucleases.

In 1955, Richards [l] described the isolation of ‘an active intermediate produced during the digestion of ribonuclease by subtilisin’. The characterisation and separation of the non-covalently linked components was described 4 years later [2] . Ribonuclease S* possesses full enzymatic activity and the same holds for the enzyme reconstituted from S-peptide and S-protein. The involvement of S-peptide residues in the binding of S-peptide to S-protein and in the enzymatic activity of the reconstituted RNase S’ has been studied by using synthetic S-peptide analogs [3,4] the cleavage by subtilisin takes place in an external loop. Klee [5] and Gold [6] did not succeed in cleaving the RNases from rat and snapping turtle with subtilisin. In this study, we present the successful cleavage with subtilisin Carlsberg of the RNase from goat, brindled gnu, giraffe, reindeer, dromedary, and red kangaroo and the isolation of the corresponding S-peptides. Differences in the observed behaviour are compared with predicted differences in conformation.

The partial amino acid sequence of a non-histone chromosomal protein.

Abstract The amino acid sequence of the first thirty nine residues of the nonhistone chromosomal protein HMG-17 has been determined. Results presented here give a molecular weight of 11,000 for the protein. Some interesting sequence homology with the trout specific histone, histone-T, is noted.

Reinvestigation of the primary structures of red deer and roe deer pancreatic ribonuclease and proline sites in mammalian ribonucleases.

The sequences of amino acid residues 15-23 of red deer (Cervus elaphus) and roe deer (Capreolus capreolus) pancreatic ribonuclease and the identity of residue 99 in roe deer ribonuclease are corrected. Earlier results are explained by the cleavage of an Asp-Pro bond in both enzymes during the treatment with CNBr in 70% formic acid and by wrong interpretations of amino acid analyses. Proline residues, which occur at a number of positions in several mammalian ribonucleases, can be accommodated in a model of bovine ribonuclease S without disrupting the conformation of the main chain.

Primary Structure of Pronghorn Pancreatic Ribonuclease: Close Relationship between Giraffe and Pronghorn

SummaryPancreatic ribonuclease from pronghorn (Antilocapra americana) was isolated and its amino acid sequence was determined from a tryptic digest of the performic acid-oxidized protein. Peptides were positioned by homology with other ribonucleases. Only peptides that differed in amino acid composition from the corresponding peptides of ox or goat ribonucleases were sequenced.In a most parsimonious tree of pancreatic ribonucleases, pronghorn and giraffe were placed together and these two were placed with the bovids, leaving the deer as a taxon separate from the other ruminants. The amino acid replacements that determine this tree topology are three rarely occurring replacements shared by pronghorn and giraffe. Notwithstanding their close phylogenetic relationship, both ribonucleases differ strongly in extent of glycosidation, net charge and antigenic properties.

The Determination of DNA Sequences

Unlike the determination of the amino acid sequence of proteins and peptides, which is based on the sequential degradation of these structures and the subsequent identification of the cleaved-off amino acid residue, DNA sequence analysis is based on high-resolution electrophoresis on denaturing polyacrylamide gels of oligonucleotides with one common end, and varying in length by a single nucleotide at the other end. Although there are several rapid methods available today for DNA sequence analysis, they all rely on high-resolution gel electrophoresis. The main difference between the methods currently used lies in the way in which the set of oligonucleotides to be separated are produced. There are two approaches to obtaining such a set of oligonucleotides. Sanger and his collaborates have developed various enzymatic methods for the determination of the sequence of nucleotides in DNA.1,2 In their procedure a DNA strand to be sequenced is used as a template for E. coli DNA polymerase I, and a short complementary fragment is used as a primer. The primer is annealed to the template and then extended enzymatically for an average of 15 to 300 or more nucleotides in the presence of radioactive labelled deoxyribonucleoside triphosphates.