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Biomedical subjects

B D Davis

Publications and source records attributed to B D Davis.

At least 91 records · Page 5Linked to original sources
PubMed1978

Interaction of secreted nascent chains with surrounding membrane in Bacillus subtilis.

To determine the length of secreted nascent polypeptide chain that is surrounded by membrane, we digested labeled nascent chains protruding from protoplasts of Bacillus subtilis with Pronase and isolated the residual ribosome-attached chains from the membrane-polysome fraction. Gel chromatography revealed a sharp major peak that had been protected by membrane plus bound ribosomes. The ribosomes themselves protected half as great a length. Because no free chain between the ribosome and the membrane was detected by Pronase treatment, the difference between the two protected lengths should measure the length protected by the membrane. More accurate measurements of these lengths, obtained by dansylation of the exposed NH2 terminus of the isolated fragments, yielded a difference of 21 amino acids. This value corresponds to an extended chain of 75 A, which is approximately the thickness of the bacterial cell membrane. We earlier presented evidence that bacterial ribosomes are attached to membrane solely by their secreted chain. The present results further show that after loss of the extracellular segment of the chain its attachment persists, at 37 degrees as well as 0 degrees C. These findings suggest that the chain does not slip through a passive membrane but is actively held within a channel.

Amino Acid Sequence↗
PubMed1978

Nascent peptide as sole attachment of polysomes to membranes in bacteria.

When membrane-polysome complexes from Escherichia coli were treated with puromycin, at various Mg2+ and K+ concentrations, the bulk of the ribosomes were released from the membrane. Moreover, many released ribosomes released ribosomes remained attached to mRNA (pseudopolysomes). These results suggest that ribosomes are attached to the membrane in bacteria solely by their nascent chain. Without a direct attachment the conformational changes associated with chain elongation on the ribosome cannot force the growing chain through the membrane, and so alternative sources of energy for the transfer must be considered.

Bacteria↗
PubMed1977

Extracellular labeling of nascent polypeptides traversing the membrane of Escherichia coli.

To provide direct evidence for the hypothesis that secreted proteins may traverse membranes as growing chains, we labeled spheroplasts of Escherichia coli with a reagent (acetyl[35S]methionyl methylphosphate sulfone) that reacts with amino groups but does not cross the membrane. After fractionation, about 6% of the label in the membrane-polysome fraction was found to be attached to the polysomes. This attachment was via peptidyl-tRNA, as shown by several tests: release of most of the label from purified polysomes at low Mg2+; subsequent loss of about 25,000 daltons on cleavage by dilute alkali; release by puromycin; and release, accompanied by a marked increase in average molecular weight, on peptide chain completion. Moreover, a significant fraction of the completed chains was identified serologically and by molecular weight as a major periplasmic protein, alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1]. This work provides direct evidence that: (i) secreted proteins thread through the membrane as growing peptide chains; and (ii) membrane-associated polysomes in bacteria are functionally attached to membrane and not merely trapped on disruption of the cell.

Alkaline Phosphatase↗
PubMed1977

Occurrence of alpha-amylase in the axis of germinating peas.

alpha-Amylase was found in the axis portion of ungerminated pea seeds (Pisum sativum var. Alaska). The occurrence of this enzyme was demonstrated with crude homogenates (also containing beta-amylase) using three different methods: the hydrolysis of beta-limit dextrin, the change in absorption spectra for the iodine-starch complex, and the increase in reducing materials relative to the decrease in starch. The first method was used to quantitate the changes in alpha-amylase activity during germination. The increase in total amylase activity (primarily beta-amylase) paralleled germination; the accumulation of alpha-amylase activity was not initiated for an additional day. The increased alpha-amylase activity was related to epicotyl growth. Approximately half of this activity was found in the etiolated stem, the distribution being higher in growing than in nongrowing portions.

Journal Article↗
PubMed1976

Susceptibility of beta-hemolytic streptococci to 65 antibacterial agents.

Tests for susceptibility of 29 group A, 4 group C, and 2 group G strains of beta-hemolytic streptococci to 63 antibiotics and to trimethoprim and sulfamethoxazole, singly and combined in a ratio of 1:16, were carried out in vitro. All strains tested were moderately or highly susceptible to all the antibiotics used except those belonging to the aminoglycoside and polymyxin groups. A few were also resistant to the tetracyclines and to sulfamethoxazole alone. Comparisons with results obtained in previous years indicate that, except for the tetracyclines and sulfonamides, there has been no change in the susceptibility of beta-hemolytic streptococci to the most important and useful antibiotics, particularly penicillin.

Aminoglycosides↗
PubMed1975

The irreversible step in formation of initiation complexes of Escherichia coli.

At some stage during initiation the ribosomal subunits of Escherichia coli must become irreversibly coupled, since polysomal ribosomes, in contrast to free ribosomes, are not dissociated by initiation factor IF-3. To determine when irreversibility develops we have compared the response to IF-3 of mature, puromycin-reactive initiation complexes, made with GTP, and of intermediate, puromycin-unreactive complexes, made with GMPPCP. The latter complexes initially appeared to be dissociated by the factor but this effect was found to be due to artificial loss of the ligands at the Mg2+ concentration customary in the test for dissociation. At a slightly higher Mg2+ concentration (4 mM), sufficient to retain the ligands, the GMPPCP complexes were not significantly dissociated by IF-3, at concentrations that caused complex dissociation of free ribosomes. It thus appears that the intermediate 70S initiation complex, though less stable to ionic dissociation than the mature complex, is in effect irreversible under physiological conditions.

Binding Sites↗
PubMed1975

Mechanism of dissociation of ribosomes of Escherichia coli by initiation factor IF-3.

Initiation factor IF-3 clearly can cause dissociation of ribosomes by binding to 30S subunits and thus shifting the spontaneous equilibrium, but it theoretically may also do so by binding to 70S ribosomes. Previously reported experiments with differentially labeled ribosomes and subunits seemed to eliminate the latter mechanism, but these experiments neglected a substantial decrease in the specific activity of the added subunits resulting from net dissociation of the ribosomes by IF-3. The present experiments eliminate or correct this effect. The results exclude direct action on 70S ribosomes as a significant mechanism of dissociation by IF-3.

Escherichia coli↗
PubMed1974

Use of purified polysomes from rabbit reticulocytes in a specific test for initiation factors.

Initiation factors, extracted from ribosomal particles by high salt, are usually tested for with the extracted ribosomes. However, it is theoretically possible that such tests may respond not only to factors (i.e., proteins that join and leave a ribosome during its cycle), but also to true ribosomal proteins that may have been extracted. We have eliminated this circularity by tests with purified polysomes, which have not been extracted with high salt; the polysomes are deficient in initiation factors because they have been largely separated from the native subunits. Tests with both systems, applied to extracts of different classes of particles, suggest that high salt does extract both initiation factors and essential ribosomal proteins. Extracts of native subunits contain both, and stimulate both systems; while, in the presence of these extracts, the extracts of polysomes further stimulate only the extracted ribosomes.

Amino Acids↗
PubMed1974

Localization of the stringent protein of Escherichia coli on the 50S ribosomal subunit.

The "stringent" protein of the ribosome, required for its synthesis of (p)ppGpp, is readily lost during zonal centrifugation. However, enough can be retained to permit its qualitative localization. It is found in native 50S subunits, runoff 70S ribosomes, and polysomes, but not in native 30S subunits. This protein, therefore, appears to be attached to the 50S moiety of the ribosome, and it may be a constant (though easily removed) component of that structure rather than a factor that joins and leaves during the ribosome cycle.

Bacterial Proteins↗