Biomedical subjects

V L Davidson

Publications and source records attributed to V L Davidson.

100 records · Page 6Linked to original sources

PubMed1985

An inducible periplasmic blue copper protein from Paracoccus denitrificans. Purification, properties, and physiological role.

When grown on methylamine as a sole carbon source, Paracoccus denitrificans synthesizes a Type I blue copper protein which mediates electron transfer between methylamine dehydrogenase and cytochrome c. This blue copper protein does not serve as an electron acceptor for methanol dehydrogenase and is not synthesized by cells grown on methanol or succinate. The blue copper protein and methylamine dehydrogenase were localized in the periplasm of P. denitrificans, whereas formate dehydrogenase was cytoplasmic. The copper protein can be purified to high yield in a single step from the periplasmic subcellular fraction prepared from P. denitrificans. The purified protein contains a single 15,000-Da polypeptide chain and one copper atom/molecule and exhibits a pI of 4.8. The oxidized form of the protein absorbs strongly at 595 nm and weakly at 464 nm. The physical and physiological properties of this protein indicate that it is not an azurin, but representative of another class of blue copper proteins.

Bacterial Proteins↗

PubMed1985

The biosynthesis and assembly of methanol dehydrogenase in bacterium W3A1.

Bacterium W3A1, a restricted facultative methylotroph, produces a periplasmic methanol dehydrogenase composed of two identical subunits of Mr = 57,300, and two noncovalently bound methoxatin prosthetic groups. A precursor form of Mr = 1,500 larger than the mature subunit was identified among the products of an in vitro translation of total RNA isolated from bacterium W3A1. The precursor form of the protein could not be detected in cells during in vivo pulse-labeling studies, suggesting that the processing of this precursor occurs entirely co-translationally. Whereas the holoenzyme was detectable only as a dimer, removal of the prosthetic group yielded an apoenzyme that could be detected as either a dimeric or monomeric species. After readdition of the purified prosthetic group to the apoenzyme, only the dimeric form of the protein, bearing the cofactor and exhibiting an absorption spectrum similar to that of the holoenzyme, was detected. Neither the mature apoprotein nor the holoenzyme demonstrated any affinity for phospholipid membranes, as assayed by their inability to bind to liposomes. Taken together, these data suggest a scheme of co-translational processing and export of the apoprotein subunits, followed by assembly of the subunits and prosthetic groups in the periplasmic space to form the mature holoenzyme. The suitability of bacterium W3A1, and other methylotrophic bacteria, for use in studies of protein biosynthesis and export, is also discussed.

Alcohol Oxidoreductases↗

PubMed1985

Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: relevance to the mechanism of action of colicins and certain toxins.

The channel-forming activity of colicin E1 in artificial membranes is known to increase at low pH values and to have a maximum near pH 4 in such membrane vesicles. The present work demonstrates that this pH dependence of activity can be attributed to membrane binding. Maximal binding of colicin E1 and a more slowly binding channel-forming carboxyl-terminal tryptic peptide occurred at acidic pH values, with the effective pK values for binding equal to 4.6 and less than 4.0, respectively. The binding did not require imposition of a transmembrane potential. Insertion of the tryptic peptide into the membrane was shown by retention of bound [3H]leucine-labeled peptide by vesicles after digestion with protease, as well as by retention of the peptide in salt-washed vesicles. The retention after protease treatment was also used to estimate the amount of carboxyl-terminal peptide inserted into the membrane. Approximately 12 of the 21 leucines present in the carboxyl-terminal peptide were retained after Pronase treatment at pH less than 4. Reversibility of the insertion at low pH values was seen after an alkaline shift of pH to 6.0, resulting in a decrease of the protease-inaccessible fraction of the bound protein. A model is presented describing a mechanism in which protonation of one or more carboxyl residues is necessary for effective binding and insertion into the membrane by the channel-forming domain of colicin E1. This model may also be relevant to the mechanism of membrane insertion by certain toxins.

Colicins↗

PubMed1985

Localization of the immunity protein-reactive domain in unmodified and chemically modified COOH-terminal peptides of colicin E1.

The region of the colicin E1 polypeptide that interacts with immunity protein has been localized to a 168-residue COOH-terminal peptide. This is the length of a proteolytically generated peptide fragment of colicin E1 against which imm+ function can be demonstrated in osmotically shocked cells. The role of particular amino acids of the COOH-terminal peptide in the expression of the immune phenotype was studied. Chemical modification showed that the two histidine residues (His 427 and His 440) and the single cysteine residue (Cys 505) present in the COOH-terminal peptide were not necessary for the colicin-immunity protein interaction. The immunity protein was localized in the cytoplasmic membrane fraction, consistent with previous work of others on the colicin Ia immunity protein and the prediction from the immunity protein amino acid sequence that it is a hydrophobic protein. The distribution of hydrophobic residues along the immunity polypeptide was calculated.

Amino Acid Sequence↗

PubMed1985

Regulation by carbon source of enzyme expression and slime production in bacterium W3A1.

Slime production by bacterium W3A1 was greatly enhanced during growth on methanol and, to a lesser extent, during growth on trimethylamine. Of the major dehydrogenases synthesized, trimethylamine and methylamine dehydrogenases were induced to different levels by certain carbon sources, while methanol dehydrogenase was expressed during growth on all carbon sources.

Alcohol Oxidoreductases↗

PubMed1984

Dependence of the activity of colicin E1 in artificial membrane vesicles on pH, membrane potential, and vesicle size.

The dependence on pH and membrane potential of the ability of colicin E1 and a COOH-terminal tryptic fragment of the colicin to form membrane channels has been measured using a chloride-sensitive electrode to measure colicin-induced ion efflux from asolectin vesicles of two different size classes. This method allows measurement of ion efflux on a faster time scale, with half-times for efflux less than or equal to 3 s, than previously possible using labeled solutes. Activity measurements were also made through the use of potential-indicating fluorescence probes. The activities of both colicin E1 and the fragment increased with decreasing pH. The activity of the colicin was maximum at pH values near 4.0, with an apparent pK of 4.5-4.6, whereas that of the COOH-terminal fragment continued to increase to the lowest pH value, 3.4, that could be used, showing an apparent pK less than or equal to 3.8. Using relatively small vesicles (average diameter approximately equal to 0.1 micron) made by a freeze-thaw procedure, chloride efflux caused by addition of fragment or colicin was independent of the initial transmembrane K+-diffusion potential imposed upon the system. However, with larger (0.5-micron diameter) vesicles prepared by a fusion method, the chloride efflux showed a dependence upon membrane potential, with the activity decreasing as the membrane potential was made more positive. The average size of the different vesicle populations was determined by electron microscopy. It is proposed that the lack of potential dependence observed in the freeze-thaw vesicles and the small voltage dependence, relative to planar membranes, seen in the larger fused vesicles, results from rapid discharge of the membrane potential and internal ion content of the vesicles.

Amino Acid Sequence↗

PubMed1984