Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Location and regulation of expression

Acinetobacter calcoaceticus possesses an L(+)-lactate dehydrogenase and a D(-)-lactate dehydrogenase. Results of experiments in which enzyme activities were measured after growth of bacteria in different media indicated that the two enzymes were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate. The two lactate dehydrogenases have very similar properties to L(+)-mandelate dehydrogenase and D(-)-mandelate dehydrogenase. All four enzymes are NAD(P)-independent and were found to be integral components of the cytoplasmic membrane. The enzymes could be solubilized in active form by detergents; Triton X-100 or Lubrol PX were particularly effective D(-)-Lactate dehydrogenase and D(-)-mandelate dehydrogenase could be selectively solubilized by the ionic detergents cholate, deoxycholate and sodium dodecyl sulphate.

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Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties

Biochemical Journal ◽

10.1042/bj2310407 ◽

1985 ◽

Vol 231 (2) ◽

pp. 407-416 ◽

Cited By ~ 11

Author(s):

N Allison ◽

M J O'Donnell ◽

C A Fewson

Keyword(s):

Lactate Dehydrogenase ◽

Ion Exchange ◽

Gel Filtration ◽

Acinetobacter Calcoaceticus ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Purification And Properties ◽

Molecular Masses ◽

Membrane Bound ◽

Lactate Dehydrogenases

Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.

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Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Blood ◽

10.1182/blood.v77.3.508.508 ◽

1991 ◽

Vol 77 (3) ◽

pp. 508-514 ◽

Cited By ~ 7

Author(s):

EI Peerschke

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Adenosine Diphosphate ◽

Time Dependent ◽

Binding Studies ◽

Fibrinogen Binding ◽

Human Thrombin ◽

Membrane Bound ◽

Independent Association ◽

Triton X

Abstract Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

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Solubilization and immunoprecipitation of 125I-labelled antigens from Plasmodium knowlesi schizont-infected erythrocytes using non-ionic, anionic and zwitterionic detergents

Parasitology ◽

10.1017/s0031182000054317 ◽

1984 ◽

Vol 88 (1) ◽

pp. 27-36 ◽

Cited By ~ 6

Author(s):

R. J. Howard ◽

J. W. Barnwell

Keyword(s):

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Plasmodium Knowlesi ◽

Polyacrylamide Gel Electrophoresis ◽

Protein A ◽

Sodium Dodecyl ◽

Ionic Detergents ◽

Class A ◽

Anionic Detergents ◽

Triton X

SUMMARYPlasmodium knowlesi malaria-infected erythrocytes were radio-iodinated and several non-ionic, anionic and zwitterionic detergents were compared in their capacity to extract the labelled membrane proteins. The use of these detergents for antigen identification was tested by immunoprecipitation, after addition of Triton X-100 to some detergent extracts, using hyperimmune monkey antiserum and protein A-Sepharose. 125I-labelled antigens were specifically immunoprecipitated with all detergents tested, including the anionic detergents sodium dodecyl sulphate (SDS), deoxycholate and cholate; the zwitterions Zwittergent-312 and -314, CHAPS and Empigen BB, as well as several non-ionic detergents. The SDS-polyacrylamide gel electrophoresis patterns of 125I-labelled antigens varied after extraction with different detergents, there being no consistent pattern for detergents of a particular class. A total of 14 125I-labelled antigens were identified, 11 of them using Triton X-100. Some minor antigens identified with Triton X-100 were immunoprecipitated in greater amount after extraction in other detergents. Most importantly, two antigens Mr 200000 and 180000 were detected only after extraction with deoxycholate or SDS.

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Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus

Biochemical Journal ◽

10.1042/bj2480871 ◽

1987 ◽

Vol 248 (3) ◽

pp. 871-876 ◽

Cited By ~ 5

Author(s):

M E Hoey ◽

N Allison ◽

A J Scott ◽

C A Fewson

Keyword(s):

Lactate Dehydrogenase ◽

Ion Exchange ◽

Gel Filtration ◽

Acinetobacter Calcoaceticus ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Ph Optimum ◽

Purification And Properties ◽

Potential Inhibitors ◽

Triton X

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

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Some observations on the choice of detergent for solubilization of the human erythrocyte membrane

Biochemical Journal ◽

10.1042/bj1640465 ◽

1977 ◽

Vol 164 (2) ◽

pp. 465-468 ◽

Cited By ~ 28

Author(s):

D A W Grant ◽

S Hjertén

Keyword(s):

Erythrocyte Membrane ◽

Sodium Dodecyl Sulphate ◽

Human Erythrocyte ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Human Erythrocyte Membrane ◽

Ionic Detergents ◽

Triton X

Solubilization of the human erythrocyte membrane by seven detergents is described. Components released into the supernatant or retained in the residue were identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two non-ionic detergents exhibiting little u.v. absorption were more efficient than u.v.-absorbing Triton X-100. Evidence is presented of an interchange between protein PAS 1 and protein PAS 2.

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Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Blood ◽

10.1182/blood.v77.3.508.bloodjournal773508 ◽

1991 ◽

Vol 77 (3) ◽

pp. 508-514

Author(s):

EI Peerschke

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Adenosine Diphosphate ◽

Time Dependent ◽

Binding Studies ◽

Fibrinogen Binding ◽

Human Thrombin ◽

Membrane Bound ◽

Independent Association ◽

Triton X

Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

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Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation

Blood ◽

10.1182/blood.v69.2.537.bloodjournal692537 ◽

1987 ◽

Vol 69 (2) ◽

pp. 537-545 ◽

Cited By ~ 1

Author(s):

JE Fox ◽

CC Reynolds ◽

JS Morrow ◽

DR Phillips

Keyword(s):

Plasma Membrane ◽

Platelet Activation ◽

Actin Filaments ◽

Sodium Dodecyl ◽

Actin Binding ◽

Ionophore A23187 ◽

Unidentified Component ◽

Membrane Bound ◽

A Minor ◽

Triton X

We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)- polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X- 100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta- aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), or N- ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.

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The Respiratory System and Diazotrophic Activity ofAcetobacter diazotrophicus PAL5

Journal of Bacteriology ◽

10.1128/jb.181.22.6987-6995.1999 ◽

1999 ◽

Vol 181 (22) ◽

pp. 6987-6995 ◽

Cited By ~ 40

Author(s):

M. Flores-Encarnación ◽

M. Contreras-Zentella ◽

L. Soto-Urzua ◽

G. R. Aguilar ◽

B. E. Baca ◽

...

Keyword(s):

Respiratory System ◽

Glucose Dehydrogenase ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Chromatography Analysis ◽

Highly Active ◽

Type Activity ◽

Membrane Bound ◽

A Minor ◽

Triton X

ABSTRACT The characteristics of the respiratory system of Acetobacter diazotrophicus PAL5 were investigated. Increasing aeration (from 0.5 to 4.0 liters of air min−1 liter of medium−1) had a strong positive effect on growth and on the diazotrophic activity of cultures. Cells obtained from well-aerated and diazotrophically active cultures possessed a highly active, membrane-bound electron transport system with dehydrogenases for NADH, glucose, and acetaldehyde as the main electron donors. Ethanol, succinate, and gluconate were also oxidized but to only a minor extent. Terminal cytochrome c oxidase-type activity was poor as measured by reducedN,N,N,N′-tetramethyl-p-phenylenediamine, but quinol oxidase-type activity, as measured by 2,3,5,6-tetrachloro-1,4-benzenediol, was high. Spectral and high-pressure liquid chromatography analysis of membranes revealed the presence of cytochrome ba as a putative oxidase in cells obtained from diazotrophically active cultures. Cells were also rich inc-type cytochromes; four bands of high molecular mass (i.e., 67, 56, 52, and 45 kDa) were revealed by a peroxidase activity stain in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. KCN inhibition curves of respiratory oxidase activities were biphasic, with a highly resistant component. Treatment of membranes with 0.2% Triton X-100 solubilized c-type cytochromes and resulted in a preparation that was significantly more sensitive to cyanide. Repression of diazotrophic activity in well-aerated cultures by 40 mM (NH4)2SO4 caused a significant decrease of the respiratory activities. It is noteworthy that the levels of glucose dehydrogenase and putative oxidaseba decreased 6.8- and 10-fold, respectively. In these cells, a bd-type cytochrome seems to be the major terminal oxidase. Thus, it would seem that glucose dehydrogenase and cytochrome ba are key components of the respiratory system of A. diazotrophicus during aerobic diazotrophy.

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Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation

Blood ◽

10.1182/blood.v69.2.537.537 ◽

1987 ◽

Vol 69 (2) ◽

pp. 537-545 ◽

Cited By ~ 93

Author(s):

JE Fox ◽

CC Reynolds ◽

JS Morrow ◽

DR Phillips

Keyword(s):

Plasma Membrane ◽

Platelet Activation ◽

Actin Filaments ◽

Sodium Dodecyl ◽

Actin Binding ◽

Ionophore A23187 ◽

Unidentified Component ◽

Membrane Bound ◽

A Minor ◽

Triton X

Abstract We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)- polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X- 100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta- aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), or N- ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.

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SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF HUMAN AND PORCINE THYROTROPHIN RECEPTORS

Journal of Endocrinology ◽

10.1677/joe.0.0780089 ◽

1978 ◽

Vol 78 (1) ◽

pp. 89-102 ◽

Cited By ~ 29

Author(s):

P. J. D. DAWES ◽

V. B. PETERSEN ◽

B. REES SMITH ◽

R. HALL

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Binding Activity ◽

Human Thyroid ◽

Scatchard Analysis ◽

Binding Characteristics ◽

Approximate Molecular Weight ◽

Membrane Bound ◽

Triton X

Thyrotrophin (TSH) receptors have been extracted from human and porcine thyroid membranes by treatment with Triton X-100.125I-Labelled bovine TSH was used to monitor receptor activity. Analysis by gel filtration and electrophoresis on acrylamide gels containing sodium dodecyl sulphate suggested that Triton extracts of human thyroid membranes contained TSH receptors with a molecular weight in the region of 50 000 closely associated with Triton micelles of approximate molecular weight 300 000. Isoelectric focusing studies indicated that the Triton-solubilized TSH binding activity had an isoelectric point of pH 4–4·5. The soluble TSH receptors were heat-labile, showed optimum TSH binding at pH 7·4 and reduced hormone binding at high ionic strength. The TSH binding characteristics of membrane-bound and solubilized human TSH receptors were similar and both preparations gave curved Scatchard plots. Solubilized porcine TSH receptors appeared to have a similar molecular weight to the human receptors and were also closely associated with Triton micelles of approximate molecular weight 300 000. Scatchard analysis of TSH binding to membrane-bound or solubilized porcine TSH receptors gave approximately linear plots with association constants of 2·8 ± 0·95 (s.e.m.) × 109 and 1·7 ± 0·27 × 1091/mol respectively. Comparison of the binding capacities of the solubilized and membrane-bound porcine receptors indicated that the 0·5% Triton extracts contained 40% of the original TSH binding activity and that this was present at a concentration of 25 ng/ml.

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