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.

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.

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

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 of Streptomyces griseus protease B, a major component of pronase

1974 ◽  
Vol 61 (4) ◽  
pp. 1095-1100 ◽  
Author(s):  
L. Jurášek ◽  
M.R. Carpenter ◽  
L.B. Smillie ◽  
A. Gertler ◽  
S. Levy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Streptomyces Griseus ◽  
Protease B

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.

scholarly journals Polysaccharide Lyase: Molecular Cloning, Sequencing, and Overexpression of the Xanthan Lyase Gene of Bacillus sp. Strain GL1

2001 ◽  
Vol 67 (2) ◽  
pp. 713-720 ◽  
Author(s):  
Wataru Hashimoto ◽  
Hikaru Miki ◽  
Noriaki Tsuchiya ◽  
Hirokazu Nankai ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Signal Peptide ◽  
Streptomyces Griseus ◽  
Peptide Sequence ◽  
Terminal Amino Acid ◽  
Signal Peptide Sequence ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

ABSTRACT When grown on xanthan as a carbon source, the bacteriumBacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520–2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus(identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4°C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) fromBacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.

scholarly journals Vaccine Potential of the Neisseria meningitidis 2086 Lipoprotein

2004 ◽  
Vol 72 (4) ◽  
pp. 2088-2100 ◽  
Author(s):  
Leah D. Fletcher ◽  
Liesel Bernfield ◽  
Vicki Barniak ◽  
John E. Farley ◽  
Alan Howell ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Neisseria Meningitidis ◽  
Bactericidal Activity ◽  
Vaccine Development ◽  
Outer Membrane Proteins ◽  
Complex Mixture ◽  
Amino Acid Sequence Similarity ◽  
Screening Methods ◽  
Protein Variant

ABSTRACT A novel antigen that induces cross-reactive bactericidal antibodies against a number of Neisseria meningitidis strains is described. This antigen, a ∼28-kDa lipoprotein called LP2086, was first observed within a complex mixture of soluble outer membrane proteins (sOMPs) following a series of fractionation, protein purification, and proteomics steps. Approximately 95 different neisserial isolates tested positive by Western blotting and PCR screening methods for the presence of the protein and the gene encoding LP2086. The strains tested included isolates of N. meningitidis serogroups A, B, C, W135, and Y, Neisseria gonorrhoeae, and Neisseria lactamica. To better understand the microheterogeneity of this protein, the 2086 genes from 63 neisserial isolates were sequenced. Two different subfamilies of LP2086 were identified based on deduced amino acid sequence homology. A high degree of amino acid sequence similarity exists within each 2086 subfamily. The highest degree of genetic diversity was seen between the two subfamilies which share approximately 60 to 75% homology at the nucleic acid level. Flow cytometry (fluorescence-activated cell sorting) analyses and electron microscopy indicated that the LP2086 is localized on the outer surface of N. meningitidis. Antiserum produced against a single protein variant was capable of eliciting bactericidal activity against strains expressing different serosubtype antigens. Combining one recombinant lipidated 2086 (rLP2086) variant from each subfamily with two rPorA variants elicited bactericidal activity against all strains tested. The rLP2086 family of antigens are candidates worthy of further vaccine development.

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