scholarly journals Immunological analysis of plant mitochondrial NADH dehydrogenases

1986 ◽  
Vol 236 (1) ◽  
pp. 201-207 ◽  
Author(s):  
I R Cottingham ◽  
M W J Cleeter ◽  
C I Ragan ◽  
A L Moore
Keyword(s):  
Nadh Dehydrogenase ◽  
Plant Mitochondria ◽  
The Other ◽  
Mitochondrial Inner Membrane ◽  
Crossed Immunoelectrophoresis ◽  
Specific Antisera ◽  
Molecular Masses ◽  
Nadh Dehydrogenases ◽  
Single Polypeptide ◽  
Triton X

Plant mitochondrial NADH dehydrogenases were analysed by two immunological strategies. The first exploited an antiserum raised to a preparation of SDS-solubilized mitochondrial-inner-membrane particles. By using a combination of activity-immunoprecipitation and crossed immunoelectrophoresis, it was shown that Triton X-100-solubilized membranes contain at least three immunologically distinct NADH dehydrogenases. Two of these were subsequently isolated by line immunoelectrophoresis and analysed for polypeptide composition: one contained three polypeptides with molecular masses of 75, 62 and 41 kDa; the other was a single polypeptide with a molecular mass of 53 kDa. The other approach was to probe plant mitochondrial membranes with antibodies raised to a purified preparation of ox heart rotenone-sensitive NADH dehydrogenase and subunits thereof. Cross-reactions were observed with the subunit-specific antisera against the 30 and 49 kDa ox heart proteins. However, the molecular masses of the equivalent polypeptides in plant mitochondria are slightly lower, at 27 and 46 kDa respectively.

scholarly journals Proteins of the kidney microvillar membrane. The amphipathic forms of endopeptidase purified from pig kidneys

1983 ◽  
Vol 211 (3) ◽  
pp. 743-753 ◽  
Author(s):  
I S Fulcher ◽  
A J Kenny
Keyword(s):  
Chelating Agents ◽  
Gel Filtration ◽  
Small Region ◽  
The Other ◽  
Kidney Cortex ◽  
Dimeric Molecule ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Hydrolysis Of ◽  
Positively Charged

The purification of detergent-solubilized kidney microvillar endopeptidase (EC 3.4.24.11) by immuno-adsorbent chromatography is described. The product (the d-form) was 270-fold purified compared with the homogenate of kidney cortex and was obtained in a yield of 5%. It was free of other peptidase activities and homogeneous by electrophoretic analyses. It contained about 15% carbohydrate and one Zn atom/subunit. Two trypsin-treated forms were also characterized. One (dt-form) was obtained by treatment of the d-form. The other (tt-form) was the result of solubilizing the membrane by treatment with toluene and trypsin. All three forms had apparent subunit Mr values of approx. 89 000, but the d-form appeared to be slightly larger than the other two. Estimates of Mr by gel filtration showed that of the tt-form to be 216 000 whereas those of the other forms were 320 000. An estimate of the detergent (Triton X-100) bound to the d- and dt-forms accounted for this difference. By several criteria, including charge-shift crossed immunoelectrophoresis and hydrophobic chromatography, the d- and dt-forms were shown to be amphipathic molecules. In contrast, the tt-form was hydrophilic in its properties. Differences in ionic properties were also noted, consistent with the loss, in the case of the dt-form, of a positively charged peptide. The results indicate that the native endopeptidase is a dimeric molecule, each subunit being anchored in the membrane by a relatively small region of the polypeptide close to one or other terminus. The d- and dt-forms had similar enzyme activity when assayed by the hydrolysis of 125I-insulin B-chain. Chelating agents and phosphoramidon inhibited the endopeptidase. The kinetic constants were determined by a new two-stage fluorimetric assay using glutarylglycylglycylphenylalanine 2-naphthylamide as substrate and aminopeptidase N (EC 3.4.11.2) to hydrolyse phenylalanine 2-naphthylamide. The Km was 68 microM and Vmax. 484nmol X min-1 X (mg of protein)-1.

scholarly journals Analysis of NADH dehydrogenases from plant [mung bean (Phaseolus aureus)] mitochondrial membranes on non-denaturing polyacrylamide gels and purification of complex I by band excision

1988 ◽  
Vol 254 (1) ◽  
pp. 303-305 ◽  
Author(s):  
I R Cottingham ◽  
A L Moore
Keyword(s):  
Gel Electrophoresis ◽  
Mung Bean ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Polyacrylamide Gels ◽  
Phaseolus Aureus ◽  
Nadh Dehydrogenases ◽  
Triton X

The present paper describes the analysis of plant mitochondrial NADH dehydrogenases using a recently developed non-dissociating gradient polyacrylamide-gel-electrophoresis technique [Kuonen, Roberts & Cottingham (1986) Anal. Biochem. 153, 221-226]. Solubilized mung-bean (Phaseolus aureus) submitochondrial particles were analysed on 3-22% (w/v) gradient polyacrylamide gels containing 0.1% Triton X-100 and stained for multiple NADH dehydrogenase activities. A rotenone-sensitive NADH dehydrogenase (Complex I) was identified on the basis of co-migration with the purified mammalian enzyme. The polypeptide composition of the plant enzyme was further analysed by band excision and SDS/polyacrylamide-gel electrophoresis.

scholarly journals Cloning of Mannose 6-phosphate Reductase from Celery.

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 898F-898
Author(s):  
John Everard ◽  
Rebecca Grumet ◽  
Wayne Loescher
Keyword(s):  
Leaf Age ◽  
Stress Factors ◽  
Induced Expression ◽  
Specific Antisera ◽  
Molecular Masses ◽  
Internal Peptide ◽  
Mannose 6 Phosphate ◽  
Single Polypeptide

In celery, photosynthetic carbon partitioning between mannitol and sucrose is highly dependent on developmental (leaf age) and environmental (salt stress) factors. Mannose 6-phosphate reductase (M6PR) mediates a key step in mannitol biosynthesis and may regulate partitioning between sucrose and mannitol. We have constructed a cDNA library and have isolated M6PR-specific clones. Before library construction, poly(A)+ RNA, extracted from newly fully expanded leaves, was translated in vitro. A single polypeptide (35.1 kD), immunoprecipitated with M6PR-specific antisera, accounted for ≈5% of the total 35S incorporated into TCA-precipitated products. Parity between the molecular masses of the immunoprecipitated product and authentic M6PR indicated minimal posttranslational modification. The unidirectional primary library, constructed in UniZap XR vector (Stratagene), consisted of 1.53 million plaque forming-units (pfus) of which <0.4% were nonrecombinant, as estimated by “blue/white”' screening. After a single amplification, ≈0.14% of the 200,000 pfus screened with M6PR-specific antisera were identified as putative M6PR clones. Following two further rounds of screening and in vivo excision of the pBluescript phagemids their identity as full length M6PR clones was confirmed as follows: 1) IPTG-induced expression of M6PR activity in crude extracts; 2) IPTG-induced expression of a polypeptide that specifically interacted with M6PR antisera and with identical mobility (on SDS gels) to authentic M6PR; 3) 100% sequence homology to an internal peptide from a tryptic digest of purified M6PR. Based on these criteria, we conclude that we successfully cloned M6PR. The sequence is similar to several reductases from both plants and animals including an aldose 6-phosphate reductase from apple. Supported by USDA-NRI grant 940-1439.

scholarly journals Regulation of malate oxidation in plant mitochondria. Response to rotenone and exogenous NAD+

1982 ◽  
Vol 208 (3) ◽  
pp. 703-711 ◽  
Author(s):  
J M Palmer ◽  
J P Schwitzguébel ◽  
I M Møller
Keyword(s):  
Nadh Dehydrogenase ◽  
Tricarboxylic Acid Cycle ◽  
Plant Mitochondria ◽  
Jerusalem Artichoke ◽  
Atp Synthesis ◽  
Matrix Space ◽  
Matrix Surface ◽  
The Matrix ◽  
Nadh Dehydrogenases ◽  
Malate Oxidation

Exogenous NAD+ stimulated the rotenone-resistant oxidation of all the NAD+-linked tricarboxylic acid-cycle substrates in mitochondria from Jerusalem artichoke (Helianthus tuberosus L.) tubers. The stimulation was not removed by the addition of EGTA, which is known to inhibit the oxidation of exogenous NADH. It is therefore concluded that added NAD+ gains access to the matrix space and stimulates oxidation by the rotenone-resistant NADH dehydrogenase located on the matrix surface of the inner membrane. Added NAD+ stimulated the activity of malic enzyme and displaced the equilibrium of malate dehydrogenase; both observations are consistent with entry of NAD+ into the matrix space. Analysis of products of malate oxidation showed that rotenone-resistant oxygen uptake only occurred when the concentration of oxaloacetate was low and that of NADH was high. Thus it is proposed that the concentration of NADH regulates the activity of the two internal NADH dehydrogenases. Evidence is presented to suggest that the rotenone-resistant NADH dehydrogenase is engaged under conditions of high phosphorylation potential, which restricts electron flux through the rotenone-sensitive dehydrogenase (coupled to ATP synthesis).

Immunoelectrophoretic Analysis of Membrane Glycoproteins in Cultured Human Endothelial Cells

1990 ◽  
Vol 63 (02) ◽  
pp. 303-311
Author(s):  
Tone Børsum
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Rabbit Antiserum ◽  
Platelet Glycoprotein ◽  
Membrane Glycoproteins ◽  
Human Endothelial Cells ◽  
Immunoelectrophoretic Analysis ◽  
Glycoprotein Complex ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryHuman endothelial cells isolated from umbilical cordswere solubilized in Triton X-100 and examined by crossedimmunoelec-trophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelialcell proteins with 14Cmannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques.Two monoclonal antibodies against the platelet glycoprotein complex Ilb-IIIa and glycoprotein IlIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex Ilb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Calmodulin-microtubule association in cultured mammalian cells.

1984 ◽  
Vol 98 (3) ◽  
pp. 904-910 ◽  
Author(s):  
W J Deery ◽  
A R Means ◽  
B R Brinkley
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell System ◽  
The Other ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Hypotonic Treatment ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Ca Sensitivity

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

Evaluation of dialysis treatment in uremic patients by gel filtration of serum

1990 ◽  
Vol 36 (11) ◽  
pp. 1906-1910 ◽  
Author(s):  
J Osada ◽  
T Gea ◽  
C Sanz ◽  
I Millan ◽  
J Botella
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Other ◽  
Control Group ◽  
Dialysis Treatment ◽  
Additional Information ◽  
Molecular Masses ◽  
Two Peaks

Abstract A group of substances of molecular masses between 300 and 1500 Da have been found to be toxic metabolites in patients with uremia. We determined the concentration in serum of these molecules in the following groups of patients: two hemodialyzed groups (one with cuprophane and the other with polyacrylonitrile dialyzers), one group treated with continuous ambulatory peritoneal dialysis, one group of nondialyzed azotemic patients, and one control group of healthy persons. Ultrafiltrates of the subjects' sera were fractionated on Sephadex G-15 followed by ion-exchange chromatography. Eluates were monitored by absorbance at 254 and 206 nm. Partially characterized peaks P1 and P2, obtained by gel filtration, correlated with the concentration of creatinine in serum; their concentrations were significantly (P less than 0.01) larger in hemodialyzed groups than in peritoneal dialyzed or in nondialyzed azotemic patients. After ion-exchange chromatography, two peaks (P'5 and P'6) correlated with serum creatinine and also were larger in hemodialyzed patients than in the other groups. Apparently, adequate discrimination is obtained by gel-filtration analysis and further analysis by ion-exchange chromatography does not provide additional information in most of the affected patients.

Sign in / Sign up

Export Citation Format

Share Document