The isolation of porcine ribonuclease, a glycoprotein, from pancreatic juice (original) (raw)
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Structure and enzymic activity of ribonuclease-A esterified at glutamic acid-49 and aspartic acid-53
Biochemical Journal, 1978
The dimethyl ester of bovine pancreatic ribonuclease-A (dimethyl RNAase-A), the initial product of esterification of RNAase-A in anhydrous methanolic HCl, was isolated in a homogeneous form. The two carboxy functions esterified in this derivative are those of glutamic acid-49 and aspartic acid-53. There were no changes in the u.v.-absorption spectral characteristics, the accessibility of the methionine residues, the resistance of the protein to proteolysis by trypsin and the antigenic behaviour of RNAase-A as a result of the esterification of these two carboxy groups. Dimethyl RNAase-A exhibited only 65% of the specific activity of RNAase-A, but still had the same Km value for both RNA and 2′:3′-cyclic CMP. However, the Vmax. was decreased by about 35%. On careful hydrolysis of the methyl ester groups at pH9.5, dimethyl RNAase-A was converted back into RNAase-A. Limited proteolysis of dimethyl RNAase-A by subtilisin resulted in the formation of an active RNAase-S-type derivative, na...
International Journal of Biochemistry
Porcine pancreatic hydrolases in juice and homogenate surveyed by electrophoretic separation in agarose gel, at pH 8.6 and subsequently characterized using substrates of various specificity, either directly in the gel or after transfer to nitrocellulose (enzymoblotting) showed: Anodal and cathodal trypsin with Bz-Arg-pNA. Chymotrypsin A, B, and C with similar, but not identical, activities to Suc-Ala-Ala-Pro-Phe-pNA, Bz-Tyr-pNA, Suc-Phe-pNA and Ac-Phe-beta NE and with differences in their molecular weights and electrophoretical charges. Elastase I and protease E with Suc-(Ala)3-pNA and MeO-Suc-Ala-Ala-Pro-Val-pNA and elastase I also with elastin. Elastase II with the chymotrypsin substrates and with elastin. Carboxypeptidase A with CN-Phe. Amylase with blue starch polymer.
Ribonucleases from rat and bovine liver: purification, specificity and structural characterization
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1998
The presence of four members of the pyrimidine-specific ribonuclease superfamily was demonstrated in rat liver. Three Ž. of them RL1, RL2 and RL3 were purified and showed ribonuclease activity at pH 7.5 with yeast RNA as substrate. RL1 is Ž. identical to rat pancreatic ribonuclease ribonuclease 1. N-terminal sequence analysis showed the presence of the native protein and several N-terminally degraded components. RL2 and RL3 were N-terminally blocked proteins. After acidic cleavage or CNBr digestion, several parts of their sequences were determined. RL2 has high sequence similarity with Ž. neurotoxin-type ribonucleases ribonucleases 2, 3 and 6. The amino acid sequence of rat liver-type ribonuclease Ž. ribonuclease 4 was determined from a liver cDNA library. It differs at about 20% of the amino acid positions from other mammalian liver-type ribonucleases. The sequence of a peptide of RL3 was identical to that derived from the cDNA sequence of the liver-type ribonuclease. A contaminant of the RL3 fraction had a high sequence similarity with mouse and Ž. other mammalian angiogenins. Bovine, porcine and rat liver-type ribonucleases showed a strong preference for poly U over Ž. poly C. This preference is a unique property of the liver-type enzymes of the ribonuclease superfamily. q 1998 Elsevier Science B.V.
Glycoconjugate Journal, 1992
The development of methods to separate, analyse and monitor changes in glycoform populations is essential if a more detailed understanding of the structure, function and processing of glycoproteins is to emerge. In this study, intact ribonuclease B was resolved by borate capillary electrophoresis into five populations according to the particular oligomannose structure associated with each glycoform. The relative proportions of these populations are correlated with the percentages obtained indirectly by analysis of the hydrazine released oligosaccharides using Bio-Gel P-4 gel filtration, matrix assisted laser desorption mass spectrometry and high performance anion exchange chromatography. Alterations in the composition of the glycoform populations during digestion of ribonuclease B with A. saitoi ~(1-2)mannosidase were monitored by capillary electrophoresis (CE). Digestion of the free oligosaccharides under the same conditions, monitored by anion exchange chromatography, revealed a difference in rate, allowing some insight into the role of the protein during oligosaccharide processing. In conjunction with other methods, this novel application of CE may prove a useful addition to the techniques available for the study of glycoform populations.
Terminal residues of hog pancreatic amylase
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1976
Studies on the identification of the terminal residues in protease-free hog pancreatic a-amylase, prepared by glycogen precipitation, demonstrated the absence of free amino terminals when four different chemical procedures were used. These methods were based on reaction with fluorodinitrobenzene, trinitrobenzenesulfonic acid, dansyl chloride, and cyanate. In the search for the presence of a possible aN blocking group, an acetyl group was detected as acetic acid dinitrophenyl hydrazide after hydrazinolysis and dinitrophenylation. Quantitation of acetyl groups by a gas chromatographic or a specific enzymatic method yielded 0.7 mol of acetyl group per 51 000 g of protein. Other acyl groups, such as formyl or propionyl, were not found. Leucine was shown to be the carboxyl terminal residue by hydrazinolysis or by carboxypeptidase A digestion of acid denatured amylase. With either procedure, 0.8 tool of carboxyl terminal leucine was found per 51 000 g of protein. These findings are consistent with the proposal that hog pancreatic a-amylase is composed of a single, a-Nacetylated chain of molecular weight 50 000. Claims of other investigators for suhunit and multichain structures for this enzyme are discussed in view of these end group data.
Affinity Chromatography of Bovine Pancratic Ribonuclease A
European Journal of Biochemistry, 1969
5'-(4-Aminophenyl-phosphoryl)-uridine-2'(3')-phosphate was synthesized and coupled to Sepharose by activation with cyanogen bromide. This conjugate was used as a specific adsorbent for purification of ribonuclease A by affinity chromatography. Totally reduced and oxidized RNase preparations which are enzymatically inactive were not adsorbed or retarded by the RNase-specific column, while S-protein is strongly retarded.