scholarly journals The amino acid sequence around the reactive serine residue of some proteolytic enzymes

1960 ◽  
Vol 77 (1) ◽  
pp. 149-163 ◽  
Author(s):  
M. A. Naughton ◽  
F. Sanger ◽  
B. S. Hartley ◽  
D. C. Shaw
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Serine Residue

scholarly journals Factor D̄ of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site

1980 ◽  
Vol 187 (3) ◽  
pp. 863-874 ◽  
Author(s):  
D M Johnson ◽  
J Gagnon ◽  
K B Reid
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Alternative Pathway ◽  
Amino Acid Sequences ◽  
Serine Residue ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat ‘group-specific protease’ [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined.

The amino acid sequence of yeast phosphoglycerate mutase

1982 ◽  
Vol 215 (1198) ◽  
pp. 19-44 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Phosphoglycerate Mutase ◽  
Crystallographic Structure ◽  
X Ray ◽  
C Terminus ◽  
Detailed Interpretation ◽  
High Degree

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8─12 residue peptide from the C-terminus.

Studies on the High-Sulfur Proteins of Reduced Merino Wool

1969 ◽  
Vol 39 (10) ◽  
pp. 917-929 ◽  
Author(s):  
T. Haylett ◽  
L. S. Swart
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Deae Cellulose ◽  
Notable Feature ◽  
Merino Wool ◽  
Amino Terminal ◽  
Sulfur Protein ◽  
Low Sulfur

The first complete amino-acid sequence of a wool protein is presented. The high-sulfur protein SCMKB-IIIB2, with a molecular weight of 11,260, consists of 97 residues and has an acetylated amino terminal. A notable feature of the protein is that it has a high- and a low-sulfur region. The sequence was determined by examination of the peptides released by various proteolytic enzymes and separated by chromatography on DEAE-cellulose with volatile buffers.

Amino acid sequence in the region of the reactive serine residue of eel acetylcholinesterase

Biochemistry ◽  
1973 ◽  
Vol 12 (15) ◽  
pp. 2946-2950 ◽  
Author(s):  
Norwood K. Schaffer ◽  
Harry O. Michel ◽  
Alec F. Bridges
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Serine Residue

An Improved Fractionation System for Pronase on CM-Sephadex

1971 ◽  
Vol 49 (11) ◽  
pp. 1195-1201 ◽  
Author(s):  
L. Jurášek ◽  
P. Johnson ◽  
R. W. Olafson ◽  
L. B. Smillie
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Complex Mixture ◽  
Streptomyces Griseus ◽  
Preparative Scale ◽  
Linear Gradient ◽  
Pyridine Acetate ◽  
Single Column ◽  
Caseinolytic Activity

An efficient, single-column preparative-scale fractionation of pronase on CM-Sephadex using a linear gradient of pyridine-acetate, pH 5.0, has been developed. Under these conditions excellent resolution and minimal autolysis of the component proteolytic enzymes occurs. Only three major endopeptidases with caseinolytic activity are found. Streptomyces griseus trypsin (S.G.T.) is recovered in adequate purity for amino acid sequence studies. Streptomyces griseus Protease A and Protease B have been shown to correspond to PNPase I and II previously described by Wählby. Two aminopeptidases and a carboxypeptidase have also been demonstrated. Pronase appears to be a less complex mixture of proteolytic enzymes than had previously been appreciated.

Amino Acid Sequence Studies of Horseradish Peroxidase. II. Thermolytic Peptides

1972 ◽  
Vol 50 (1) ◽  
pp. 63-90 ◽  
Author(s):  
K. G. Welinder ◽  
L. B. Smillie
Keyword(s):  
Amino Acid ◽  
Horseradish Peroxidase ◽  
Amino Acid Sequence ◽  
Cation Exchanger ◽  
Paper Electrophoresis ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Disulfide Bridges ◽  
Serine Residue ◽  
C Terminus

Horseradish peroxidase (HRP) was digested with thermolysin. On fractionation on Sephadex G-25, Fine Chromobeads type P (Dowex 50 type resin) and by high-voltage paper electrophoresis, we isolated about 120 thermolytic peptides. Some experimentation on the composition of the pyridine acetate gradient, used for elution of the cation exchanger, is reported. All peptides were characterized with respect to amino acid composition, N-terminal residue, and pH 6.5 mobility. Unknown peptides or peptides not corresponding unambiguously to previously established tryptic sequences were subjected to dansyl-Edman analysis. Thermolytic peptides accounting for all tryptic sequences except a dipeptide and a tripeptide, and unique thermolytic sequences accounting for about 100 amino acid residues, were obtained. Nine convincing and several indicative overlaps were established for known tryptic sequences. The sequences around all four disulfide bridges, the three histidine residues, and the only tryptophan residue have been elucidated. Eight sites of carbohydrate attachment have been identified. For seven of these sites we have evidence for attachment to asparagine, and for six of the sites the carbohydrate-bound asparagine was found in the well-known sequences Asn–X–Ser/Thr. The remaining two sequences, though incomplete, are compatible with this pattern. Tentatively we suggest a pyrrolidone carboxyl N-terminal for HRP. The specificity of trypsin implicates a sequence found in two varieties, differing only by a C-terminal serine residue at the C-terminus of HRP. A discussion of the possible complications of the acidic heme extraction on the results obtained is included.

scholarly journals Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

2002 ◽  
Vol 68 (7) ◽  
pp. 3532-3536 ◽  
Author(s):  
María J. Benito ◽  
Mar Rodríguez ◽  
Félix Núñez ◽  
Miguel A. Asensio ◽  
María E. Bermúdez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Penicillium Chrysogenum ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Extracellular Protease ◽  
Collagenolytic Activity ◽  
Sds Page

ABSTRACT An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

Amino acid sequence of cyanogen bromide fragments CB4 and CB6 of hog pepsin

1981 ◽  
Vol 46 (3) ◽  
pp. 626-639
Author(s):  
Ladislav Morávek
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Disulfide Bond ◽  
Sequential Analysis ◽  
Cyanogen Bromide ◽  
Serine Residue ◽  
Aspartic Acid Residue ◽  
N Terminus ◽  
Methionine Residues ◽  
Cyanogen Bromide Fragment

By the analyses of chymotryptic, and thermolytic peptides the amino acid sequence was determined of cyanogen bromide fragment CB4 representing the region of the pepsin chain between the N-terminus and methonine-residue I: Ile-Gly-Asp-Glu-Pro-Leu-Glu-Asn-Tyr-Leu-Asp-Thr-Glu-Tyr-Phe-Gly-Thr-Ile-Gly-Ile-Gly-Thr-Pro-Ala-Gln-Asp-Phe-Thr-Val-Ile-Phe-Asp-Thr-Gly-Ser-Ser-Asn-Leu-Trp-Val-Pro-Ser-Val-Tyr-Cys-Ser-Ser-Leu-Ala-Cys-Ser-Asp-His-Asn- + Gln-Phe-Asn-Pro-Asp-Asp-Ser-Ser-Thr-Phe-Glu-Ala-Thr-Ser-Gln-Glu-Leu-Ser-Ile-Thr-Tyr-Gly- + Thr-Gly-Ser-Met. The serine residue (Ser) in position 68 of pepsin is phosphorylated. By sequential analysis of chymotryptic, tryptic, and thermolytic peptides the amino acid sequence was determined of cyanogen bromide fragment CB6 representing the region between methionine residues II and III in the pepsin chain: Asp-Gly-Glu-Thr-Ile-Ala-Cys-Ser-Gly-Gly-Cys-Gln-Ala- + Ile-Val-Asp-Thr-Gly-Thr-Ser-Leu-Leu-Thr-Gly-Pro-Thr-Ser-Ala-Ile-Ala-Asn-Ile-Gln-Ser-Asp- + Ile-Gly-Ala-Ser-Glu-Asn-Ser-Asp-Gly-Glu-Met. The aspartic acid residue (Asp) in position 16 of this fragment is identical with the residue reacting with diazo inhibitors which forms a part of the active center of the enzyme. Both half-cystine residues of fragment CB4 and fragment CB6 are linked to one another by a disulfide bond in native pepsin.

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