scholarly journals The mother-cell-membrane adenosine triphosphatase of sporulating Clostridium pasteurianum

1980 ◽  
Vol 186 (1) ◽  
pp. 191-199 ◽  
Author(s):  
David J. Clarke ◽  
J. Gareth Morris
Keyword(s):  
Cell Membrane ◽  
Mother Cell ◽  
Vegetative Cell ◽  
Sodium Dodecyl ◽  
Proton Channel ◽  
Clostridium Pasteurianum ◽  
Synthetase Activity ◽  
Inhibitor Protein ◽  
Protein Subunit ◽  
Membrane Atpase

1. Sporulation of Clostridium pasteurianum effects several changes in its proton-translocating cell-membrane H+-ATPase. Notable among these are the acquisition of susceptibility to activation by trypsin and a changed protein subunit composition. 2. A protein was isolated from the mother-cell membrane that inhibited the ATP phosphohydrolase activity of purified vegetative-cell-membrane H+-ATPase [BF0F1 complex, which consists of soluble ATPase (BF1) and the proton-channel component (BF0)] and rendered it susceptible to trypsin activation. 3. This trypsin-sensitive inhibitor protein had a molecular weight of 10000 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was indistinguishable from the novel protein subunit e of the mother-cell-membrane ATPase 4. In bacteriorhodopsin-containing everted membrane vesicles, the specific ATP synthetase activity of the mother-cell-membrane ATPase was significantly greater than that of the vegetative-cell-membrane ATPase. 5. Treatment with trypsin-sensitive inhibitor protein of artificial proteoliposomes containing bacteriorhodopsin and vegetative-cell-membrane H+-ATPase (BF0F1) significantly increased the specific ATP synthetase activity of this enzyme. 6. The ATP synthetase activity of crude cell-membrane preparations from cultures of Clostridium pasteurianum increased during that period in the course of sporulation when the membrane ATP phosphohydrolase was both most rapidly decreasing in specific activity and acquiring its susceptibility to activation by trypsin.

scholarly journals Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic BacteriumPhotorhabdus luminescens

2001 ◽  
Vol 67 (10) ◽  
pp. 4834-4841 ◽  
Author(s):  
David J. Bowen ◽  
Jerald C. Ensign
Keyword(s):  
Exponential Growth ◽  
Galleria Mellonella ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Cellular Protein ◽  
Cross Reactivity ◽  
Crystalline Inclusion ◽  
Protein Subunit ◽  
Inclusion Type ◽  
Isolation And Characterization

ABSTRACT Cells of the entomopathogenic bacterium Photorhabdus luminescens contain two types of morphologically distinct crystalline inclusion proteins. The larger rectangular inclusion (type 1) and a smaller bipyramid-shaped inclusion (type 2) were purified from cell lysates by differential centrifugation and isopycnic density gradient centrifugation. Both structures are composed of protein and are readily soluble at pH 11 and 4 in 1% sodium dodecyl sulfate (SDS) and in 8 M urea. Electrophoretic analysis reveals that each inclusion is composed of a single protein subunit with a molecular mass of 11,000 Da. The proteins differ in amino acid composition, protease digestion pattern, and immunological cross-reactivity. The protein inclusions are first visible in the cells at the time of late exponential growth. Western blot analyses showed that the proteins appeared in cells during mid- to late exponential growth. When at maximum size in stationary-phase cells, the proteins constitute 40% of the total cellular protein. The protein inclusions are not used during long-term starvation of the cells and were not toxic when injected into or fed toGalleria mellonella larvae.

scholarly journals Dibutylchloromethyltin chloride, a covalent inhibitor of the adenosine triphosphate synthase complex

1977 ◽  
Vol 166 (3) ◽  
pp. 593-602 ◽  
Author(s):  
Kelvin Cain ◽  
Michael D. Partis ◽  
David E. Griffiths
Keyword(s):  
Oxidative Phosphorylation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Interaction ◽  
Sodium Dodecyl ◽  
Mitochondrial Atpase ◽  
Protein Subunit ◽  
Binding Studies ◽  
Mitochondrial Atp Synthase ◽  
Chloroform Methanol ◽  
Covalent Inhibitor

1. The synthesis of dibutylchloromethyltin chloride, a new covalent inhibitor of the mitochondrial ATP synthase [oligomycin-sensitive ATPase (adenosine triphosphatase)] complex is described, together with a method for preparing dibutylchloro[3H]methyltin chloride. 2. Studies with the yeast mitochondrial oligomycin-sensitive ATPase complex show that dibutylchloromethyltin chloride inhibits both the membrane-bound enzyme and also the purified Triton X-100-dispersed preparation. 3. F1-ATPase is not inhibited even at 500nmol of dibutylchloromethyltin chloride/mg of protein, and the general inhibitory properties are similar to those of triethyltin, oligomycin and dicyclohexylcarbodi-imide, known energy-transfer inhibitors of oxidative phosphorylation. 4. Binding studies with yeast submitochondrial particles show that dibutylchloromethyltin chloride antagonizes the binding of triethyl[113Sn]tin, indicating that there is an interaction between the two inhibitor-binding sites. 5. Unlike triethyltin, inhibition by dibutylchloromethyltin chloride is due to a covalent interaction which titrates a component of the inner mitochondrial membrane present at a concentration of 8–9nmol/mg of protein. 6. All of the labelled component can be extracted with chloroform/methanol (2:1, v/v), and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chloroform/methanol extract indicates that the labelled component has an apparent mol.wt. of 6000–8000. However, t.l.c. reveals the presence of only one labelled component which is lipophilic and non-protein and is distinct from the free inhibitor, mitochondrial phospholipids and the dicyclohexylcarbodi-imide-binding protein (subunit 9). 7. Inhibition of mitochondrial ATPase and oxidative phosphorylation is correlated with specific interaction with a non-protein lipophilic component of the mitochondrial inner membrane which is proposed to be a co-factor or intermediate of oxidative phosphorylation.

Development of sperm cells in barley

1975 ◽  
Vol 53 (10) ◽  
pp. 1051-1062 ◽  
Author(s):  
David D. Cass ◽  
Ilana Karas
Keyword(s):  
Cell Membrane ◽  
Vegetative Cell ◽  
Generative Cell ◽  
Pollen Grains ◽  
Complete Separation ◽  
Pollen Wall ◽  
Vegetative Nucleus ◽  
Sperm Cells ◽  
Subsequent Stage ◽  
Microspore Stage

Ultrastructural events in barley sperm development were examined from the uninucleate microspore stage to establishment of two mature sperm cells in pollen grains. Microspore mitosis produces a vegetative nucleus and a naked generative cell, both embedded in vegetative cell cytoplasm. The generative cell membrane is enclosed by vegetative cell membrane. The generative cell, at first apparently unattached, becomes attached to the pollen wall and acquires a cell wall by centripetal vesicle accumulation. Wall formation may be complete at the time of generative cell karyokinesis; karyokinesis occurs while the generative cell is attached to the pollen wall. Cytokinesis of the generative cell is delayed. The subsequent stage is a binucleate, attached generative cell with a wall. Generative cell cytokinesis appears to involve formation of a partition between the two sperm nuclei. Eventual complete separation of the sperm cells occurs only after the two-celled derivative of the generative cell detaches from the pollen wall. Final stages in sperm cell separation are considered to result from degradation of the partitioning and surrounding wall, not from furrowing of a naked binucleate generative cell according to previous suggestions. Mature plastids were not observed in the generative cell or the sperms.

Effect of Sulfur Supply on the Seed Globulin Composition of Various Species of Lupin

1978 ◽  
Vol 5 (5) ◽  
pp. 641 ◽  
Author(s):  
JM Gillespie ◽  
RJ Blagrove ◽  
PJ Randall
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Large Change ◽  
Subunit Composition ◽  
Sulfur Deficiency ◽  
Protein Subunit ◽  
Disulfide Bonding ◽  
Molecular Weights ◽  
Sulfur Supply ◽  
The Individual

The protein level in seeds of six species of lupin, grown either under sulfur deficiency or with adequate sulfur fertilization, is marginally affected by sulfur supply. However, the ratio of total sulfur to total nitrogen in the seed is greatly decreased under sulfur deficiency. This large change in sulfur-to-nitrogen ratio is accompanied by suppression of the synthesis of conglutins α and γ, which contain a significant amount of cyst(e)ine and methionine. The level of protein is maintained by increased synthesis of conglutin β, which normally contains no methionine and a low proportion of cyst(e)ine. These changes in the proportions of the proteins are reflected in the amino acid analyses for the globulin extracts. The changes in protein subunit composition which accompany the differences in the proportions of the proteins have been studied using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The results emphasize the differences in subunit composition between lupin species in terms of the number of components, their molecular weights and the importance of disulfide bonding. Two-dimensional electrophoresis, using cellulose acetate and SDS-polyacrylamide gradient gels, has been used to compare the subunit composition of the individual globulins for Lupinus angustifolius and L. elegans at both sulfur levels.

scholarly journals Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane

1985 ◽  
Vol 227 (2) ◽  
pp. 413-420 ◽  
Author(s):  
Y Kajita ◽  
C R Rickards ◽  
P R Buckland ◽  
R D Howells ◽  
B Rees Smith
Keyword(s):  
Cell Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Fat Cell ◽  
Disulphide Bridge ◽  
B Subunit ◽  
Hydrophilic Peptide ◽  
A Subunit

Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.

Purification of the heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase by affinity chromatography

1978 ◽  
Vol 56 (6) ◽  
pp. 598-604 ◽  
Author(s):  
Rajendra K. Sharma ◽  
Rekha Desai ◽  
Thomas R. Thompson ◽  
Jerry H. Wang
Keyword(s):  
Molecular Weight ◽  
Protein Kinase ◽  
Cyclic Nucleotide ◽  
Sodium Dodecyl ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Brain Extract ◽  
Affinity Column ◽  
Inhibitor Protein ◽  
Camp Phosphodiesterase ◽  
Heat Stable

The recently discovered heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase (Sharma, R, K., Wirch, E. &Warg, J. H. (1978) J. Biol. Chem., in press) has been purified 238 214-fold from bovine brain extract using an affinity column of the modulator protein – Sepharose 4B conjugate. The purified sample appears to be homogeneous as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The protein band has a mobility corresponding to that of a polypeptide of molecular weight 68 000. Since the heat-stable inhibitor protein has a molecular weight of 70 000 under nondenaturing conditions, it suggests that it is a monomeric protein. The protein has no inhibitory activity toward the cAMP-dependent protein kinase or protein phosphatase. The purified sample has been tested for various enzyme activities which include ATPase, GTPase, cAMP phosphodiesterase, cGMP phosphodiesterase, 5′-nucleotidase, and protein kinase. None of these activities are exhibited by the purified sample.

scholarly journals Characteristics of Drosophila rhodopsin in wild-type and norpA vision transduction mutants.

1978 ◽  
Vol 72 (5) ◽  
pp. 717-732 ◽  
Author(s):  
S E Ostroy
Keyword(s):  
Extinction Coefficient ◽  
Visual Pigment ◽  
Sodium Dodecyl ◽  
Receptor Potential ◽  
Molar Extinction Coefficient ◽  
Light Cycle ◽  
Wild Type ◽  
Protein Subunit ◽  
Spectral Identification ◽  
Protein Components

The properties of the major visual pigment of Drosophila melanogaster were evaluated. The visual pigment was isolated from other protein components using acrylamide gel electrophoresis and spectral identification. Sodium dodecyl sulfate (SDS) acrylamide gels of the isolated visual pigment gave a single protein subunit with a mol wt of 37,000 daltons. The rhodopsin480 molar extinction coefficient was 35,000 liter/mol-cm (+/- 2,700 SE). The metarhodopsin580 molar extinction coefficient was approximately 56,000 liter/mol-cm. Microspectrophotometry was used to compare the rhodopsin concentrations in wild-type flies and norpA vision transduction mutants. At 2 days of age (12 h dark-12 h light cycle, 19 degrees C) all of the norpA flies exhibited a similar rhodopsin concentration (75% of the wild-type strain). By 21 days of age some of the norpA alleles showed substantially reduced rhodopsin concentrations (16-43% of normal), whereas others showed no major age-dependent decreases (68-77%). Temperature and light-dark cycle affected the reduction. Alleles with no receptor potential exhibited the largest decreases in rhodopsin concentration. The data indicate that the norpA phototransduction mutant has a defect in the system responsible for maintaining the rhodopsin480 concentration. This defect in the rhodopsin maintenance system does not appear to be the cause of the reduced electroretinogram (ERG) amplitude observed in some of these mutants, but instead is a consequence of the decrease in ERG amplitude, or the flaw(s) responsible for the decrease in ERG amplitude.

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