PubMed Health⌕ Search

Biomedical subjects

B C Monk

Publications and source records attributed to B C Monk.

32 records · Page 2Linked to original sources
PubMed1992

The regulatory domain of fungal and plant plasma membrane H(+)-ATPase.

The activity of fungal and plant plasma membrane H(+)-ATPases seems to be regulated by modulation of the interaction of an inhibitory domain at the C-terminus with the active site. In the yeast ATPase, a mutation at the active site (Ala547- > Val) and a deletion of the C-terminus result in constitutive activation. A double Ser911- > Ala, Thr912- > Ala mutation at the C-terminus (defining putative phosphorylation sites) locks the enzyme in the inhibited state and can be suppressed by the Ala547- > Val mutation at the active site. This provides genetic evidence for domain interaction. In plant ATPase, proteolytic removal of the C-terminus also results in constitutive activation. A peptide covering a region of the plant C-terminus with homology to the yeast C-terminus inhibits the truncated plant ATPase. This suggests similar regulatory mechanisms in fungal and plant ATPases.

Cell Membrane↗
PubMed1992

Genetic probing of the yeast plasma membrane H(+)-ATPase.

The H(+)-ATPase from Saccharomyces cerevisiae has been probed by a random genetic approach that has led to the isolation of primary and secondary site mutations. These H(+)-ATPase (PMA1) mutants help define specific functional, as well as interacting, regions of the H(+)-ATPase. Cellular resistance to hygromycin B has been an important selection tool for the isolation of pmal mutants. One prominent hygromycin B-resistant mutant, pmal-105, was found to have a S368F mutation near the site of phosphorylation (D378) in the catalytic core. This mutation prevents growth in low pH or NH(4+)-containing medium and induces an acid-sensitive Vmax for ATP hydrolysis, as well as a pronounced insensitivity to vanadate. The prominent cellular and biochemical phenotypes of this strain facilitated a detailed revertant analysis to identify protein structure domains that interact directly or indirectly with the localized region defined by the F368 mutation. Partial revertants were isolated which were resistant to low pH or NH4+ but retained hygromycin resistance. Second site mutations were found within the first and second cytoplasmic loop domains, as well as in transmembrane segments 1-3 & 7. All of the revertant enzymes have a stable Vmax but some show changes in the pH optimum for ATP hydrolysis; all display vanadate sensitivities ranging between the insensitive F368 mutant and the fully-sensitive wild type enzyme. Revertant analyses have also been performed on two other pma1 mutants which carry the mutations A135V and G158D in transmembrane segments 1 and 2, respectively. Compensating second site mutations to these mutations were identified in transmembrane segments 1, 2, 4 & 7, as well as within the central catalytic domain. These analyses have helped identify interacting protein structure domains that may participate in coupling ATP hydrolysis to proton transport. Furthermore, they facilitate the construction of structural models to account for these interactions.

Cell Membrane↗
PubMed1991

Immunological approaches to the transmembrane topology and conformational changes of the carboxyl-terminal regulatory domain of yeast plasma membrane H(+)-ATPase.

Molecular genetic experiments have suggested that the carboxyl terminus of the Saccharomyces cerevisiae plasma membrane H(+)-ATPase is an inhibitory domain involved in the "in vivo" regulation of the enzyme by glucose metabolism. An antibody prepared against a fusion protein including the last 59 amino acids of the ATPase sequence has been affinity purified to yield a preparation which requires the 18 carboxyl-terminal amino acids for recognition. Antibody binding experiments show that the carboxyl-terminal domain of the ATPase can be selectively exposed by concentrations of the detergent Tween-20 which do not break down the permeability barrier of the plasma membrane to the antibody. Both enzyme-linked immunosorbent assay and immunofluorescence analysis demonstrate that the accessibility of the carboxyl-terminal domain in isolated plasma membranes depends on the physiological state of the cell being increased by glucose metabolism. Immunofluorescence analysis of isolated plasma membrane vesicles, using a dual labeling protocol with concanavalin A and antibody to reveal the orientation of individual vesicles, and colloidal gold immunoelectron microscopy of ultrathin cryosections of whole yeast cells separately demonstrate that the ATPase carboxyl terminus is located in the cytoplasmic compartment. The application of a mutant deleted of the epitope(s) recognized by the affinity purified carboxyl-terminal antibody eliminates the possibility of artifacts arising from nonspecific antibody binding. The accessibility properties and cytoplasmic location of the carboxyl-terminal domain appear to be consistent with its role as a negative regulator of the ATPase.

Blotting, Western↗
PubMed1991

Domains of yeast plasma membrane and ATPase-associated glycoprotein.

In yeast homogenates the plasma membrane H(+)-ATPase and a major surface glycoprotein of about 115 kDa are present in two membrane fractions with peak densities in sucrose gradients of 1.17 and 1.22. Immunogold electron microscopy of frozen yeast sections indicates that the ATPase is exclusively (greater than 95%) present at the surface membrane. Therefore the two ATPase-containing fractions appear to correspond to different domains of the plasma membrane. The 115 kDa glycoprotein is tightly associated with the ATPase during solubilization and purification of the enzyme. However, in a mutant lacking the glycoprotein the activity of the plasma membrane H(+)-ATPase is similar to wild type, suggesting that this association is fortuitous. The ATPase and the glycoprotein are difficult to separate by electrophoresis and therefore binding of concanavalin A to the ATPase cannot be unambiguously demonstrated in wild-type yeast. By utilizing the mutant without glycoprotein it was shown that the ATPase band of 105 kDa binds concanavalin A.

Cell Membrane↗
PubMed1991

Cloning and characterization of the plasma membrane H(+)-ATPase from Candida albicans.

The Candida albicans PMA1 gene was isolated from a genomic library by using a hybridization probe obtained from the PMA1 gene of Saccharomyces cerevisiae. The gene was localized to chromosome III of the Candida genome. An open reading frame of 2,685 nucleotides predicts an amino acid sequence of 895 amino acids that is 83% homologous at both the DNA and protein levels to its S. cerevisiae equivalent. A polyadenylated mRNA transcript of about 4,000 nucleotides contains a highly folded AU-rich leader of 242 nucleotides. The structure of the gene, codon bias, and levels of approximately 100-kDa H(+)-ATPase protein recovered in plasma membranes indicate a highly expressed gene. The plasma membrane ATPase was purified to about 90% homogeneity and appeared to be blocked at the amino terminus. Three hydrophobic membrane sector tryptic fragments from the partially digested ATPase provided internal sequence information for over 50 amino acids, which agrees with the sequence predicted by the cloned gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the C. albicans enzyme is about 3 kDa smaller than its Saccharomyces counterpart and was consistent with a predicted Mr of 97,398. Antibodies to the S. cerevisiae whole ATPase or its carboxyl terminus bound to the C. albicans enzyme but with lower avidity. Kinetic analysis showed that the Candida and Saccharomyces ATPases respond to glucose activation-starvation in nonidentical fashions. The amino-terminal domain of the C. albicans ATPase is marked by a net deletion of 23 amino acids in comparison with the S. cerevisiae ATPase. These differences maintain net charge, occur in nonconserved regions of fungal ATPases, and are sufficient to account for the observed difference in electrophoretic mobility between the two yeast ATPases.

Amino Acid Sequence↗
PubMed1990

Phenotypic analysis of bovine papillomavirus type 1 E2 repressor mutants.

The bovine papillomavirus type 1 (BPV-1) E2 open reading frame encodes three proteins: the E2 transcriptional transactivator, the E2 transcriptional repressor (E2-TR), and the E8/E2 fusion peptide. In this study, we describe the phenotypes of BPV-1 mutants which are disrupted in their capacity to encode either the E2 transcriptional repressor or the E8/E2 fusion peptide. We also describe experiments which demonstrate that the E8/E2 gene product functions similarly to E2-TR. In the context of the entire viral genome, disruption of E8/E2 expression had little effect on viral processes, whereas disruption of E2-TR expression resulted in a complex phenotype marked by a 10- to 20-fold increase in viral DNA plasmid copy number as well as increased transformation potential. A double mutant, defective in the expression of both E8/E2 and E2-TR proteins, had high levels of E2 transactivation activity yet had reduced plasmid replication capacity and a delayed capacity to transform rodent cells.

Animals↗
PubMed1989

Sidedness of yeast plasma membrane vesicles and mechanisms of activation of the ATPase by detergents.

The binding of concanavalin A and of fluorescein 5'-isothiocyanate indicate similar amount of right-side-out and inside-out vesicles in plasma membrane vesicles from either glucose-starved or glucose-fermenting yeast cells. These vesicles contain low-activity and high-activity states of the ATPase, respectively. Unmasking of latent active sites can explain the limited ATPase activation (about 2-fold) produced by several detergents on both kinds of vesicles. On the other hand, lysophosphatidic acid (oleoyl) produces a 7-fold activation of the ATPase in vesicles from glucose-starved cells. This effect is accompanied by a change in Km of the enzyme and probably reflects a direct action of the detergent on the ATPase. A similar activation and Km change can be obtained by sonication of the vesicles, although in this case soybean phospholipids are required for maximal activity. Apparently the low-activity state of the yeast plasma membrane ATPase can be activated not only by glucose metabolism 'in vivo' (mechanism unknown) but also by some detergents and physical treatments 'in vitro'. Experiments with purified ATPase from glucose-starved cells also indicate that lysophosphatidic acid (oleoyl) specifically activates the enzyme. These results suggest a note of caution on considering the usual interpretation of the effects of detergents on membrane enzymes, which only take into account the unmasking of latent active sites.

Adenosine Triphosphatases↗
PubMed1989

Laser excitation of fluorescent-labeled polypeptides in polyacrylamide gels.

A laser beam at 488 nm, converted into a fan of light by a surface-coated mirror oscillated in response to a triangular wave, was inserted into the base of a polyacrylamide gel. The laser light was trapped by internal reflection and gave uniform illumination throughout the entire gel slab. Photography with color film detected 50 fmol of fluorescein covalently coupled to ovalbumin, gave 80-fold greater sensitivity than transillumination in detection of fluorescein-labeled polypeptides, and was about 25-fold more sensitive than protein staining with silver. Laser illumination visualized end-labeled beta-galactosidase, afforded quality control of such preparations, and demonstrated that the end-labeled derivative contained about 25-fold less fluorescein than uniformly labeled beta-galactosidase. The latter result was confirmed by dot-blot analysis using a polyclonal antibody specific for fluorescein. The application of end-labeling to the location of features of protein primary structure is discussed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Characterization of the peribacteroid membrane ATPase of lupin root nodules.

Peribacteroid membranes can be isolated in essentially pure form from 20-day lupin root nodules by osmotic shock of the purified membrane enclosed bacteroids. The ATPase (EC 3.6.1.3) associated with this membrane has an acid pH optimum (5.25) and is specific for ATP (Mg-ATP Km = 0.16 mM). The enzyme activity requires magnesium or manganese ions, is slightly stimulated by the cations potassium and rubidium, and is inhibited by vanadate, diethylstilbestrol, N,N'-dicyclohexylcarbodiimide, fluoride, molybdate, and calcium. Molybdate and fluoride sensitivity do not in this case indicate the presence of significant nonspecific phosphatase activity. The ATPase is not inhibited by oligomycin, azide, or the soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some respects the lupin peribacteroid membrane ATPase appears to differ from the plasma membrane ATPase of other plants.

Adenosine Diphosphate↗
PubMed1987

Electrotransfer of SDS-PAGE separated polypeptides to the DE81 blotting matrix and detection of Chlamydomonas antigens and glycoconjugates.

Electrotransfer of SDS-PAGE-separated polypeptides to nitrocellulose is not quantitative under the conditions described by Towbin et al. (1979). The use of FITC-labelled polypeptide markers and FITC-labelled Chlamydomonas flagella has allowed investigation of separate aspects of the electrotransfer process. These aspects include electroelution from the polyacrylamide gel, binding to the blotting matrix and electrophoretic re-elution from the blotting matrix. Factors which influence electrotransfer, including electrophoretic field strength, time-dependence of electrotransfer, the effect of medium composition and the binding capacity of the DE81 blotting matrix have been examined. SDS-PAGE-separated polypeptides up to Mr 350 000 can be electrotransferred to DE81 in a nearly quantitative manner in a dilute Laemmli (1970) electrophoresis medium containing 0.05% SDS in an electric field of 4 V/cm for 4 h. The efficient electrotransfer of polypeptides over a wide Mr range has allowed a study of the cross-reactivity of polyclonal antisera raised against Chlamydomonas cell walls, isolated flagella and the flagellar 350 000 major membrane glycoprotein. The principal epitopes recognised by the cross-reactive antibodies appear to be periodate-sensitive carbohydrate residues of cell wall and flagellar glycoproteins. These epitopes do not appear to include ConA binding sites.

Antigens↗
PubMed1982

Sexual agglutinins from the Chlamydomonas flagellar membrane. Partial purification and characterization.

Chlamydomonas sexual agglutinins have been quantitatively extracted from isolated flagella in vitro using the dialyzable nonionic detergent octyl-D-glucopyranoside and from cells in vivo with 12.5 mM EDTA. Both preparations elicit normal sexual responses from gametes of complementary, but not like, mating types. Extracts of vegetative cells and several agglutination-deficient (imp) mutants are totally inactive. Agglutinin activity is sensitive to trypsin, mild periodate oxidation, and heating at 60 degrees C for 1 min. These findings, coupled with the size of the molecule (it is excluded from Sepharose 6B and sediments as a 12 S particle in sucrose gradients) lead us to propose that the Chlamydomonas sexual agglutinins are large glycoproteins or glycoprotein aggregates which associate with the flagellar membrane in an extrinsic fashion. Partial purification of in vivo 125I-surface labeled EDTA extracts rules out several surface polypeptides, including the bulk of material migrating in the region of the major membrane glycoprotein (Mr 350,000), as agglutinin candidates and indicates that the active molecule is a minor component of the flagellar membrane. In addition, in vitro assays suggest a mechanism for in vivo sexual agglutination whereby stable adhesion is achieved by the active redistribution of agglutinins to the flagellar tips.

Agglutinins↗
PubMed1975

Biogenesis of mitochondria 36, The genetic and biochemical analysis of a mitochondrially determined cold sensitive oligomycin resistant mutant of Saccharomyces cerevisiae with affected mitochondrial ATPase assembly.

The isolation and characterisation of a mutant affecting the assembly of mitochondrial ATPase is reported. The mutation confers resistance to oligomycin and venturicidin and sensitivity of growth on nonfermentable substrates to low temperature (19degrees). Genetic analysis indicates that the phenotype is due to a single mutation located on the mitochondrial DNA which is probably allelic with the independently isolated oligomycin resistance mutation [oli1-r]. Growth of the mutant at the non-restrictive temperature (28degrees) yields mitochondria in which the ATPase appears more sensitive to oligomycin than that of the sensitive parental strain. However, when the enzyme is isolated free from the influence of the membrane strong resistance to oligomycin is evident. These data suggest that the component responsible for the oligomycin resistance of the ATPase is part of or subject to interaction with the mitochondrial inner membrane. Measurements of the ATPase content of mitochondria indicate that ATPase production is impaired during growth at 19degreesC. In addition, studies of the maximum inhibition of mitochondrial ATPase activity by high concentrations of oligomycin suggest a selective lesion in ATPase assembly at low temperature. The nett result is that during growth at 19degrees only about 10% of the normal level of ATPase is produced of which less than half is membrane integrated and thus capable of oxidative energy production. We propose that the mutation affects a mitochondrially synthesised membrane sector peptide of the ATPase which defines the interaction of F1ATPase with specific environments on the mitochondrial inner membrane.

Adenosine Triphosphatases↗
PubMed1975

Biogenesis of mitochondria 40. Phenotypic suppression of a mitochondrial mutation by a nuclear gene in Saccharomyces cerevisiae.

A mutant has been isolated which carries a nuclear mutation capable of suppressing certain aspects of the phenotype imposed by a specific mitochondrial mutation. The mitochondrial mutation [tso-r] confers cold sensitivity to growth on nonfermentable substrates and resistance to oligomycin. When both the mitochondrial and nuclear mutations are present in the same cell the cell is phenotypically cold resistant but retains a high level of oligomycin resistance. The extent of cold sensitivity suppression is dependent upon other unspecified nuclear genes. The molecular basis for the suppression may involve interactions between cytoplasmic and mitochondrial ATPase.

Cold Temperature↗