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Biomedical subjects

B D Davis

Publications and source records attributed to B D Davis.

At least 73 records · Page 4Linked to original sources
PubMed1983

Association of penicillin-binding proteins and other enzymes with the ribosome-free membrane fraction of Bacillus subtilis.

We had previously separated the ribosome-complexed and -free membrane fractions of Bacillus subtilis by sedimentation in a biphasic sucrose gradient. We now have found that the complexed fraction is contaminated with ribosome-free vesicles and that these can be removed by equilibrium density centrifugation. With this improved preparation, it could be shown that the penicillin-binding proteins are present almost exclusively in the ribosome-free membrane fraction. It thus appears that the fragmentation of the membrane in the lysing protoplast yields separate vesicles for the domains involved in protein translocation and for those involved in the synthesis and reshaping of the peptidoglycan. An enzyme of lipid synthesis (phosphatidylserine synthase) and also H+-ATPase were similarly found to be concentrated, but less exclusively, in the ribosome-free membrane fraction.

Adenosine Triphosphatases↗
PubMed1983

Mechanism of protein excretion by gram-negative bacteria: Pseudomonas aeruginosa exotoxin A.

Excretion of proteins by a cell with a double membrane may involve mechanisms different from secretion across a single membrane. We studied this problem with Pseudomonas aeruginosa exotoxin A. This 68,000-dalton protein was released as rapidly as it was completed; even after short pulse-labeling the cells contained neither the toxin nor a larger precursor. Excretion is evidently cotranslational, since in fractionated lysates the toxin was formed (almost entirely in the mature form) by the membrane-polysome complexes but not by the free polysomes. When the membrane was perturbed by 10% ethanol, the cells stopped excreting the toxin and they accumulated an immunoprecipitable, enzymatically active precursor of 71,000 daltons. The precursor was located entirely in the outer membrane on its outer surface. On removal of the ethanol, the cells again excreted mature toxin, but they did not process or release the previously accumulated precursor. Based on these data, a model for the excretion of exotoxin A is presented.

ADP Ribose Transferases↗
PubMed1981

Bacillus licheniformis penicillinase: cleavages and attachment of lipid during cotranslational secretion.

The penicillinase of Bacillus licheniformis is shown to be secreted cotranslationally. In extracts it was formed by membrane-associated but not by free polysomes; and after extracellular labeling of cells, followed by completion of the growing chains on polysomes in vitro, labeled penicillinase could be immunoprecipitated. This product contained electrophoretic peaks of Mr 36,000, 33,000, and 29,000, which correspond to previously reported forms of the enzyme. The Mr 36,000 form exhibits moderate hydrophobicity, as expected of a precursor with an NH2-terminal signal sequence for secretion. In addition, part of the Mr 33,000 fraction evidently contains a lipid: it is even more hydrophobic, and [2-3H]glycerol was found to be incorporated into these molecules but not into the other forms of the enzyme. These findings renew the earlier, discarded suggestion that the Mr 33,000 membrane-bound penicillinase in the cells contains lipid. The incorporation of lipid and two different cleavages can evidently all occur during growth of the penicillinase chain. Moreover, the resulting terminal regions are all accessible to extracellular labeling on growing chains. Several additional, unidentified lipoproteins also incorporate lipid during chain growth.

Bacillus↗
PubMed1980

Frontiers of the biological sciences.

The history of the molecular revolution in biology is described, emphasizing its dependence on the emergence of bacterial genetics, the fusion of genetics and biochemistry, and the development of greatly improved techniques for studying macromolecules. Central concepts have included molecular information transfer, both by nucleic acids and by allosteric proteins; the spontaneous conversion of one-dimensional information into three-dimensional structures; and the extraordinary unity in the molecular mechanisms underlying the rich diversity of biology. The merging of molecular and morphological studies, to yield the very broad field of cell biology, is described more briefly, as are also some present frontiers in several areas of biology that present challenges at other levels of organization.

Allergy and Immunology↗
PubMed1980

The mechanism of protein secretion across membranes.

Many secreted proteins are synthesised as a large precursor with an additional hydrophobic N-terminal signal sequence that is cleaved by a membrane-bound enzyme. The proteins are secreted as nascent chains. The work leading to the current models of protein secretion is reviewed and the value of bacterial systems in the study of protein transfer across membranes is stressed.

Amino Acid Sequence↗
PubMed1980

Precursor in cotranslational secretion of diphtheria toxin.

By extracellular labeling of peptides of intact Corynebacterium diphtheriae, followed by fractionation of the cells and chain completion by isolated polysomes, it is shown that diphtheria toxin is formed and secreted cotranslationally by membrane-bound polysomes; free polysomes from none. Moreover, when the chains on these polysomes were completed in vitro, in the absence of membrane they were found to include not only diphtheria toxin of a molecular weight of 62,000, but also a larger precursor of a molecular weight of 68,000. The precursor was identified by several properties: immune precipitation; conversion into toxin fragments A and B; adenosine diphosphate ribosyl-transferase activity after activation with trypsin; and cleavage to 62,000 daltons by membrane enzymes. The precursor yields an N-terminal A fragment with a broadened molecular weight distribution, compared with that from authentic toxin, thus supporting the expectation that the extra segment of the precursor is N-terminal.

Adenosine Diphosphate Ribose↗
PubMed1979

Triphasic concentration effects of gentamicin on activity and misreading in protein synthesis.

Gentamicin is shown to exert a triphasic concentration effect on peptide synthesis in vitro with natural messengers. Low concentrations (up to 2 micron) caused slowing and a decrease in total synthesis, but little misreading (assayed with extracts lacking Glu-tRNA); the inhibition was greater with an initiating system (with phage RNA as messenger) than with pure chain elongation on purified endogenous polysomes of Escherichia coli. Moderate concentrations (up to 100 micron) slowed synthesis less, markedly increased its duration in the noninitiating system, and strongly stimulated misreading; at optimal concentrations total synthesis was even greater than normal. Moreover, with phage RNA these concentrations increased the synthesis of large polypeptides. We conclude that binding of gentamicin to its first site causes inhibition but little misreading; binding to additional site(s) partly reverses the inhibition by first-site binding and markedly stimulates misreading, and the misreading appears to favor "readthrough" of termination codons. In the third phase (greater than 100 micron) synthesis is slowed again but the pattern of misreading does not appear to be altered; this effect need not involve a specific further action on the ribosome.

Bacterial Proteins↗
PubMed1979

Extracellular labeling of growing secreted polypeptide chains in Bacillus subtilis with diazoiodosulfanilic acid.

Studies of the mechanism of protein secretion in a Gram-positive bacterium, Bacillus subtilis, yielded results very similar to those previously obtained with a Gram-negative organism: nascent chains protruding from protoplasts could be labeled extracellularly; the labeled chains could be recovered on polysomes isolated from the membrane--polysome fraction; they could be released by puromycin, low Mg2+, or chain completion; the completed chains include a known secreted protein (alpha-amylase); and their ribosomes appear to be attached to membrane solely by their nascent chains. The reagent used for extracellular labeling, [1252]diazoiodosulfanilic acid, yielded severalfold more specific labeling of the nascent chains (7--10% of the total cellular labeling and one-fourth to one-third of that of the membrane--polysome fraction) than was obtained earlier with another nonpenetrating reagent.

Amylases↗
PubMed1979

Influence of Cotyledons upon alpha-Amylase Activity in Pea Embryonic Axes.

alpha-Amylase activity remained relatively low in the axes of intact etiolated pea seedlings; the activity was predominantly confined to the epicotyl. Starch accumulated slightly. When the cotyledons were removed and the axes cultured on medium containing no carbon source, the starch reserve in the axes disappeared within a few days. This was accompanied by a 10- to 15-fold increase in alpha-amylase activity, in the absence of additional epicotyl growth. The phenonemon was observed for axes throughout early growth, although the relative accumulation of alpha-amylase activity in cultured axes was less for older seedlings. This change was attributed to a reduced response by nongrowing tissues. There was no corresponding change in beta-amylase activity. These observations, described for several varieties of peas, demonstrate the control of cotyledons upon the utilization of stored reserves within the axis, with alpha-amylase as a key enzyme.

Journal Article↗
PubMed1978

Streptomycin causes misreading of natural messenger by interacting with ribosomes after initiation.

The induction of misreading by streptomycin in vitro, previously observed with synthetic messengers, is now demonstrated with natural (endogenous or viral) messenger by the use of extracts of temperature sensitive mutants lacking Glu--tRNA or Val--tRNA synthetase. With chain-elongating but noninitiating ribosomes (i.e., purified polysomes) deprived of an aminoacyl--tRNA, streptomycin and other aminoglycosides, over a wide range of concentrations, stimulate incorporation. With ribosomes initiating in the presence of streptomycin stimulation is also observed but it is restricted, just like phenotypic suppression in cells, to very low streptomycin concentrattions which evidently allow some ribosomes to initiate and later encounter them in the course of chain elongation. The stimulation is accompanied by an increase in the size of the products; hence, it is evidently due to substitution of an incorrect aminoacyl--tRNA for a missing one. The test introduced here also has revealed a misreading effect of streptomycin on resistant ribosomes. In addition, significant intrinsic misreading was observed without streptomycin, indicating that under optimal conditions for in vitro protein synthesis an empty codon is frequently read by an incorrect aminoacyl--tRNA.

Anti-Bacterial Agents↗