scholarly journals The covalent nature of the human antithrombin III–thrombin bond

1980 ◽  
Vol 189 (3) ◽  
pp. 481-489 ◽  
Author(s):  
M O Longas ◽  
T H Finlay
Keyword(s):  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Disulphide Bridge ◽  
Terminal Amino Acid ◽  
Carboxypeptidase B ◽  
Terminal Amino ◽  
Covalent Nature

1. Cleavage of the human antithrombin III–thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III–thrombin complex, the inhibitor is cleaved at an arginine–X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).

scholarly journals Purification and Properties of an Esterase from the YeastSaccharomyces cerevisiae and Identification of the Encoding Gene

1999 ◽  
Vol 65 (8) ◽  
pp. 3470-3472 ◽  
Author(s):  
Giuliano Degrassi ◽  
Lasse Uotila ◽  
Raffaella Klima ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Terminal Amino Acid ◽  
Purification And Properties ◽  
Terminal Amino ◽  
Encoding Gene ◽  
Esterase Activities ◽  
Null Mutants

ABSTRACT We purified an intracellular esterase that can function as anS-formylglutathione hydrolase from the yeastSaccharomyces cerevisiae. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50°C and pH 7.0. The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized toS-formylglutathione by S. cerevisiae.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

Human Prostatic Acid Phosphatase: Properties of the Native Enzyme, and the Enzyme-Antibody Complex

1983 ◽  
Vol 20 (6) ◽  
pp. 374-380 ◽  
Author(s):  
Renze Bais ◽  
Anne Huxtable ◽  
John B Edwards
Keyword(s):  
Acid Phosphatase ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Prostatic Acid Phosphatase ◽  
Native Enzyme ◽  
Terminal Amino Acid ◽  
Large Molecular Weight ◽  
Antibody Complex ◽  
Terminal Amino

Acid phosphatase purified from human prostatic tissue was shown to be homogeneous by polyacrylamide gel electrophoresis and N-terminal amino acid analysis. However, isoelectric focusing revealed a large number of isoenzymes which were reduced to four by digestion with neuraminidase. It is suggested that the patterns observed are due to differences in bound carbohydrate attached to the same protein backbone. Antiserum to the purified enzyme was produced in rabbits and reacted with the enzyme to form an enzymatically active complex of large molecular weight. This complex is more stable at high temperatures than the native enzyme. Kinetic analysis of both the enzyme and the enzyme-antibody complex demonstrated that the binding of the antibody caused no significant change to the active site of the enzyme.

scholarly journals Expression, Secretion, and Glycosylation of the 45- and 47-kDa Glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans

2004 ◽  
Vol 70 (2) ◽  
pp. 679-685 ◽  
Author(s):  
Martha Lara ◽  
Luis Servín-González ◽  
Mahavir Singh ◽  
Carlos Moreno ◽  
Ingrid Cohen ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Recombinant Protein ◽  
Gel Electrophoresis ◽  
Molecular Mechanisms ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Streptomyces Lividans ◽  
Mass Spectrometry Analysis ◽  
Native Protein ◽  
Two Dimensional Gel Electrophoresis

ABSTRACT The gene encoding the 45/47 kDa glycoprotein (Rv1860) of Mycobacterium tuberculosis was expressed in Streptomyces lividans under its own promoter and under the thiostrepton-inducible Streptomyces promoter PtipA . The recombinant protein was released into the culture medium and, like the native protein, migrated as a double band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with concanavalin A (ConA). Carbohydrate digestion with jack bean α-d-mannosidase resulted in a reduction in the molecular mass of the recombinant protein upper band and completely eliminated ConA binding. Two-dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein. Comparative fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels could be due to differences in glycosylation. Mass spectrometry analysis of the recombinant protein indicated that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glycosylated. Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against the carbohydrate residues of the glycoprotein. Altogether, these observations show that expression of genes for mycobacterial antigens in S. lividans is very useful for elucidation of the functional role and molecular mechanisms of glycosylation in bacteria.

scholarly journals Subcellular Localization of the Intracellular Survival-Enhancing Eis Protein of Mycobacterium tuberculosis

2001 ◽  
Vol 69 (7) ◽  
pp. 4295-4302 ◽  
Author(s):  
John L. Dahl ◽  
Jun Wei ◽  
James W. Moulder ◽  
Suman Laal ◽  
Richard L. Friedman
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intracellular Survival ◽  
Terminal Amino Acid ◽  
Terminal Amino

ABSTRACT Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designatedeis, was found to enhance intracellular survival ofMycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377–384, 2000). Wheneis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eisgene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product ofeis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence presented here suggests it associates with the cell surface and is released into the culture medium. It is produced during human tuberculosis infection and therefore may be an important M. tuberculosis immunogen.

Irreversible heat denaturation of bovine α-lactalbumin

1986 ◽  
Vol 53 (2) ◽  
pp. 249-258 ◽  
Author(s):  
Lesley C. Chaplin ◽  
Richard L. J. Lyster
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heat Denaturation ◽  
Two Dimensional ◽  
Native Protein ◽  
Denatured Protein ◽  
Hydrolysis Of

SUMMARYThe irreversible heat denaturation of α-lactalbumin (α-la) in 0·1 M-phosphate, pH 7·0, at 100 °C was studied using polyacrylamide-gel electrophoresis (PAGE). PAGE revealed two groups of bands, one moving faster than native α-la and one slower, in addition to some denatured protein which remained at the origin and some residual native α-la. The faster group had unchanged molecular weight, but an increase in charge, partly due to hydrolysis of glutamine and asparagine residues. The slower group was shown by two-dimensional sodium dodecyl sulphate-PAGE to be oligomers of denatured α-la; formation of the smaller oligomers preceded the larger ones. The oligomers reverted to monomers in the presence of dithiothreitol, showing that they were disulphide-linked aggregates of denatured α-la. Immuno-blots of the gels showed that both fast and slow groups of bands had irreversibly lost most of the antigenicity of the native protein.

scholarly journals Purification and characterization of a labile rat glutathione transferase of the Mu class

1989 ◽  
Vol 260 (3) ◽  
pp. 789-793 ◽  
Author(s):  
A Kispert ◽  
D J Meyer ◽  
E Lalor ◽  
B Coles ◽  
B Ketterer
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Glutathione Transferase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Cnbr Cleavage

A labile GSH transferase homodimer termed 11-11 was purified from rat testis by GSH-agarose affinity chromatography followed by anion-exchange f.p.l.c. The enzyme is unstable in the absence of thiol(s) and has relatively low affinity for both 1-chloro-2,4-dinitrobenzene (Km 4.4 mM) and GSH (Km(app.) 4.4mM). Its mobility on SDS/polyacrylamide-gel electrophoresis is slightly less than that of subunits 3 and 4 and its pI is 5.2. Subunit 11 has a blocked N-terminal amino acid residue, but after CNBr cleavage fragments accounting for 113 amino acid residues were sequenced and showed 65% homology with corresponding sequences in subunit 4, indicating that it is a member of the Mu family. GSH transferase 11 is a major isoenzyme in testis, epididymis, prostate and brain and present at lower concentrations in other tissues.

Production, Purification, and Characterization of Micrococcin GO5, a Bacteriocin Produced by Micrococcus sp. GO5 Isolated from Kimchi

2005 ◽  
Vol 68 (1) ◽  
pp. 157-163 ◽  
Author(s):  
MI-HEE KIM ◽  
YOON-JUNG KONG ◽  
HONG BAEK ◽  
HYUNG-HWAN HYUN
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Broad Spectrum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
High Heat ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Strain GO5, a bacteriocin-producing bacterium, was isolated from green onion kimchi and identified as Micrococcus sp. The bacteriocin, micrococcin GO5, displayed a broad spectrum of inhibitory activity against a variety of pathogenic and nonpathogenic microorganisms, as tested by the spot-on-lawn method; its activity spectrum was almost identical to that of nisin. Micrococcin GO5 was inactivated by trypsin (whereas nisin was not) and was completely stable at 100°C for 30 min and in the pH range of 2.0 to 7.0. Micrococcin GO5 exhibited a typical mode of bactericidal activity against Micrococcus flavus ATCC 10240. It was purified to homogeneity through ammonium sulfate precipitation, ultrafiltration, and CM-Sepharose column chromatography. The molecular mass of micrococcin GO5 was estimated to be about 5.0 kDa by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and in situ activity assay with the indicator organism. The amino acid sequence of micrococcin GO5 lacks lanthionine and β-methyllanthionine and is rich in hydrophobic amino acids and glycine, providing the basis for the high heat stability of this bacteriocin. The N-terminal amino acid sequence of micrococcin GO5 is Lys-Lys-Ser-Phe-Cys-Gln-Lys, and no homology to bacteriocins reported previously was observed in the amino acid composition or N-terminal amino acid sequence. Based on the physicochemical properties, small molecular size, and inhibition of Listeria monocytogenes, micrococcin GO5 has been placed with the class II bacteriocins, but its broad spectrum of activity differs from that of other bacteriocins in this class.

Radioiodinated Human Fibrinogen. In Vitro Effects of Increasing Iodination

1975 ◽  
Author(s):  
B. E. Ly ◽  
P. Kierulf
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Clotting Time ◽  
Terminal Amino Acid ◽  
Human Fibrinogen ◽  
Terminal Amino ◽  
In Vitro Effects ◽  
Polypeptide Chains

Fibrinogen preparations with increasing contents of iodine, ranging from 0.2 to 20 atoms of iodine per molecule fibrinogen, were obtained with the ICl method. Aggregation and shortening of the thrombin clotting time occurred when the content of iodine exceeded 3 atoms per molecule.Upon the action of thrombin, the increase in N-terminal glycine, reflecting fibrin formation, was almost identical in native and iodinated fibrinogen. At visible gelation, however, decreased amounts of N-terminal glycine were found in heavily iodinated fibrinogen, thus indicating enhanced fibrin polymerization. N-terminal analysis of heavily iodinated fibrinogen demonstrated a deficiency in N-terminal tyrosine concomitantly with the apparance of a new N-terminal amino acid, identified as mono-iodo-tyrosine.Polyacrylamide gel electrophoresis at pH 8.9 revealed an increase in mobility following extensive iodination, but no shift in the isoelectric point was observed upon isofocusing.Neither clottability nor the behaviour of fibrinogen and its subunit polypeptide chains on SDS-gel electrophoresis was affected by iodination.

scholarly journals Purification and characterization of rat adipose tissue lipoprotein lipase

1982 ◽  
Vol 207 (3) ◽  
pp. 485-495 ◽  
Author(s):  
S M Parkin ◽  
B K Speake ◽  
D S Robinson
Keyword(s):  
Adipose Tissue ◽  
Affinity Chromatography ◽  
Lipoprotein Lipase ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sepharose 4B

Lipoprotein lipase (EC 3.1.1.34) extracted from adipose tissue of glucose-fed rats with 5 mM-sodium barbital, pH 7.5, containing 20% (v/v) glycerol and 0.1% (v/v) Triton X-100, was partially purified by affinity chromatography on heparin linked to Sepharose 4B. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the partially purified enzyme preparation revealed the presence of two major Coomassie-staining bands (mol.wts. 62 000 and 56 000) as well as a number of minor bands. Treatment of partially purified enzyme with [1,3-3H]di-isopropyl fluorophosphate resulted in the incorporation of radiolabel into the band of mol.wt. 56 000, but not into the band of mol.wt. 62 000. Both the amount of the 56 000-mol.wt. polypeptide and the incorporation of [1,3-3H]di-isopropyl fluorophosphate into this band were greatly reduced in the enzyme preparations isolated from adipose tissue of 48 h-starved rats. whereas the amount of the 62 000-mol.wt. polypeptide was unaffected by starvation. Purification of lipoprotein lipase from adipose tissue of glucose-fed rats was also carried out using affinity chromatography on Sepharose 4B linked to heparin with low affinity for antithrombin-III. This procedure resulted in the presence of a single band of mol.wt. 56 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These results suggest that the polypeptide of mol.wt. 56 000 corresponds to the subunit of lipoprotein lipase, whereas the 62 000-mol.wt. polypeptide probably represents antithrombin-III.

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