The purification and characterization of a DNA nicking–closing enzyme from Bacillus megaterium

1978 ◽  
Vol 56 (2) ◽  
pp. 123-128 ◽  
Author(s):  
M. G. Burrington ◽  
A. Richard Morgan
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Bacillus Megaterium ◽  
Enzyme Preparation ◽  
Micrococcus Luteus ◽  
Purification And Characterization ◽  
Dna Nicking ◽  
Negative Supercoils

Although several eucaryote DNA nicking–closing (N–C) enzymes have been characterized, only the Escherichia coli enzyme has been extensively studied amongst procaryotes. The latter enzyme is distinctly different from the eucaryotic enzymes and we have therefore purified the N–C enzyme from Bacillus megaterium to determine if procaryotes form a distinctive class of N–C enzymes. The purified B. megaterium N–C enzyme has a molecular weight of 120 000, only partly relaxes negative supercoils, does not affect positive supercoils, requires Mg2+, and is inhibited by 0.2 M KCl. The enzyme is also inhibited by 1 mM nalidixic or oxolinic acids but unaffected by novobiocin. A crude N–C enzyme preparation from Micrococcus luteus shows very similar properties.

scholarly journals Purification and Characterization of PBP4a, a New Low-Molecular-Weight Penicillin-Binding Protein fromBacillus subtilis

2001 ◽  
Vol 183 (5) ◽  
pp. 1595-1599 ◽  
Author(s):  
Colette Duez ◽  
Marc Vanhove ◽  
Xavier Gallet ◽  
Fabrice Bouillenne ◽  
Jean-Denis Docquier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Binding Protein ◽  
Order Rate Constant ◽  
Low Molecular Weight ◽  
Penicillin Binding Protein ◽  
Purification And Characterization ◽  
The Third

ABSTRACT Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits dd-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781–788, 1992), which is rapidly inactivated by many β-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k 2/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M−1s−1 for the Actinomadura sp. strain R39 peptidase, 1,400 M−1 s−1 for B. subtilis PBP4a, and 7,000 M−1 s−1 forEscherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k 2/K = 46,000 M−1 s−1). PBP4a is also much more thermostable than the R39 enzyme.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

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