Jj Beintema

Demonstration by mass spectrometry that pseudo-hevein and hevein have ragged C-terminal sequences☆

The primary structure of pseudo-hevein, a minor hevein component from the latex of the rubber tree, Hevea brasiliensis, was determined. Six differences with the sequence of the major hevein component were found, one of which is a replacement of tryptophan by tyrosine in the carbohydrate binding region of the molecule. Analysis by ion-spray mass spectrometry showed that pseudo-hevein has a heterogeneous C-terminal extension of several glycine residues and that hevein itself also contains minor components with additional C-terminal amino-acid residues. A seventh difference between the two sequences occurs in these extensions.

The primary structure of giraffe pancreatic ribonuclease

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Rat pancreatic ribonuclease II. Amino acid sequence

Abstract The amino acid sequence of rat pancreatic ribonuclease was determined using 120 mg of the enzyme oxidised with performic acid. Tryptic and chymotryptic peptides were obtained by gel filtration and chromatography on ion exchangers, and if necessary cleaved to smaller fragments. In the discussion of the results, emphasis is laid on specific problems encountered during this study, e.g. conversion of asparagine residues and N-terminal glutamine residues to products which interfere with the Dansyl-Edman procedure. All amino acid residues could be located unambiguously. The position of one amide group could not be established with certainty. The total sequence consists of three inner sections in addition to the N-terminal and C-terminal sections. Since the order of the three inner sections could not be determined directly, due to the lack of overlapping peptides, these three inner sections were ordered by homology with other ribonucleases. Compared with bovine ribonuclease, rat ribonuclease contains 3 amino acid residues extra at the N-terminus, and 41 substitutions in the remainder of the chain. However, all amino acids which are part of the active site, or are implicated in activity or conformation, are identical in both enzymes.

Rat pancreatic ribonuclease. I. Isolation and properties.

Abstract Rat pancreatic ribonuclease was isolated and purified by acid extraction from pancreatic tissue followed by (NH4)2SO4 fractionation and column chromatography on Sephadex G-25, carboxymethylcellulose and Amberlite CG-50. After column chromatography, more than 95% of the enzyme activity appears in one peak. Earlier eluting glycosidated components are either absent or present in an amount of 4% at most. In physicochemical behaviour, the rat enzyme is not very different from the beef enzyme; it is more soluble in (NH4)2SO4 solutions and binds stronger to cation exchangers than the bovine enzyme. At pH 7.5 the specific activity of the rat enzyme on the substrates, ribonucleic acid and cyclic 2′,3′-cytidine monophosphate is about half, and on cyclic 2′,3′-uridine monophosphate about twice that of the beef enzyme. The rat and beef enzymes have about the same molecular weight; they contain no tryptophan, but otherwise their amino acid compositions are quite different. Both contain three abnormally titrating tyrosine residues, but the number of normal residues is different, viz. 3 in the bovine and 1 in the rat enzyme. Peptide maps of both enzymes are very dissimilar.

Immunologic comparison of pancreatic ribonucleases

Abstract Ouchterlony double immunodiffusion and micro-complement fixation were used in cross-reactivity studies with 9 pancreatic ribonucleases differing 3–28% in amino acid sequence and rabbit antisera to cow, gnu, reindeer and whale ribonuclease. Generally a correlation was observed between the extent of cross-reactivity and amino acid sequence resemblance. The antiserum against whale ribonuclease however, reacted to a larger degree with several antigens than expected from the amino acid sequence difference. Dromedary ribonuclease, which differs at 26% of the positions, showed the highest cross-reaction. By comparing the antigenic reactivities and the differences in sequence, an attempt was made to localize antigenically relevant regions.

Studies on the covalent structure of eland pancreatic ribonuclease

Studies on the covalent structure of eland (Taurotragus oryx) pancreatic ribonuclease have been performed on tryptic and thermolysin digests. The first 45 residues have been determined with a Beckman sequencer. From the remaining part of the sequence only those peptides were sequenced that differed in amino acid composition with the corresponding peptide of bovine ribonuclease. Eland pancreatic ribonuclease differs in four positions from bovine pancreatic ribonuclease A, but more differences due to a different state of amidation may be present. The absence of an Asn-X-Thr/Ser sequence in the covalent structure of eland ribonuclease (asparagine 34 has been substituted by aspartic acid) explains the absence of a glycosidated component in eland ribonuclease.

Panulirus Interruptus Hemocyanin: The amino-acid sequence of the region containing one copper-binding site and the sites susceptible to limited proteolysis

The amino-acid sequence of a part of Panulirus interruptus hemocyanin subunit a comprising the three ligands of the first copper ion has been determined. By fitting this sequence to the electron density map (Gaykema W.P.J.; Hol, W.G.J.; Vereijken J.M; Soeter N.M.; Bak H.J.; Beintema J.J. (1984) Nature (Lond.) 309, 23–29), all three ligands were found to be histidines. Comparison of the sequence with other sequenced subunits of arthropodan hemocyanins showed that a part of the residues, including the liganding histidines, has been conserved during evolution. The sequenced part of the molecule also contained the cleavage sites for proteolytic enzymes, located at the surface of the native molecule.

N-terminal amino acid sequence of trypsinogen from the lesser rorqual, Balaenoptera acutorostrata (Cetacea). Simultaneous isolation of trypsinogen, chymotrypsinogen and ribonuclease from pancreas.

Abstract Ribonuclease, trypsinogen and two chymotrypsinogens have been extracted from a pancreas of a whale. The activation peptide of trypsinogen has been isolated and its sequence compared with other known sequences. The relative position of the Cetacea among the orders of Mammalia is discussed. A sequence is proposed for the common ancestor of the mammals considered.