Purification and Characterization of a High-Molecular-Weight Insecticidal Protein Complex Produced by the Entomopathogenic BacteriumPhotorhabdus luminescens

1998 ◽  
Vol 64 (8) ◽  
pp. 3029-3035 ◽  
Author(s):  
David J. Bowen ◽  
Jerald C. Ensign
Keyword(s):  
Molecular Weight ◽  
Insecticidal Activity ◽  
Protein Complex ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Toxin ◽  
The Family ◽  
Toxin Complex

ABSTRACT Photorhabdus luminescens is a gram-negative enteric bacterium that is found in association with entomopathogenic nematodes of the family Heterorhabditidae. The nematodes infect a variety of soil-dwelling insects. Upon entering an insect host, the nematode releases P. luminescens cells from its intestinal tract, and the bacteria quickly establish a lethal septicemia. When grown in peptone broth, in the absence of the nematodes, the bacteria produce a protein toxin complex that is lethal when fed to, or injected into the hemolymph of, Manduca sexta larvae and several other insect species. The toxin purified as a protein complex which has an estimated molecular weight of 1,000,000 and contains no protease, phospholipase, or hemolytic activity and only a trace of lipase activity. The purified toxin possesses insecticidal activity whether injected or given orally. Analyses of the denatured complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of several protein subunits ranging in size from 30 to 200 kDa. The complex was further separated by native gel electrophoresis into three components, two of which retained insecticidal activity. The purified native toxin complex was found to be active in nanogram concentrations against insects representing four orders of the classInsecta.

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.

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

1979 ◽  
Author(s):  
M.J. Rabiet ◽  
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-Va1-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 1 (P1) and the appearance of abnormal intermediates migra-ti ng faster than P1.

Isolation, purification, and partial characterization of type V-A hemagglutinin from Escherichia coli GV-12, O1:H-

1982 ◽  
Vol 152 (2) ◽  
pp. 757-761
Author(s):  
V L Sheladia ◽  
J P Chambers ◽  
J Guevara ◽  
D J Evans
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Amino Terminus ◽  
N Terminus ◽  
Type V

A hemagglutinin which specifically agglutinates human type A erythrocytes (mannose resistant) was isolated from the growth medium of cultures of Escherichia coli GV-12, serotype O1:H-, and purified by chromatography on Bio-Gel A-1.5 and DEAE-Sephadex A-25. The purity of the hemagglutinin was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis. N-terminus analysis indicated that only asparagine resides on the amino terminus. The native hemagglutinin is an aggregate exhibiting a sedimentation coefficient of 9.25, which corresponds to a molecular weight of approximately 200,000. The monomeric molecular weight was found to be approximately 16,300. Amino acid analysis indicated that the hemagglutinin consists of 131 residues, corresponding to a molecular weight of 13,400.

Purification and Characterization of an Unusual Aminopeptidase From Pea Seeds

1980 ◽  
Vol 7 (2) ◽  
pp. 131 ◽  
Author(s):  
JB Caldwell ◽  
LG Sparrow
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Group ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Pea Seeds ◽  
Hydrolysis Of

An aminopeptidase with specificity for N-terminal glutamic and aspartic acid residues has been purified to apparent homogeneity from pea seeds (Pisum sativum cv. Greenfeast). It also catalyses the hydrolysis of the glutaryl-phenylalanine bond of the synthetic chymotrypsin substrate glutaryl- L-phenylalanine p-nitroanilide. The native enzyme, which has a molecular weight of approximately 500 000, gives a single band on polyacrylamide gel electrophoresis but two major bands when subjected to electrophoresis in the presence of sodium dodecyl sulfate after reduction. Its behaviour with various inhibitors suggests that a sulfhydryl group is important for its activity.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

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