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Biomedical subjects

W A Cramer

Publications and source records attributed to W A Cramer.

At least 109 records · Page 6Linked to original sources
PubMed1983

Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide.

A COOH-terminal tryptic fragment (Mr approximately equal to 20,000) of colicin E1 has been proposed to contain the membrane channel-forming domain of the colicin molecule. A comparison is made of the conductance properties of colicin E1 and its COOH-terminal fragment in planar bilayer membranes. The macroscopic and single channel properties of colicin E1 and its COOH-terminal tryptic fragment are very similar, if not indistinguishable, implying that the NH2-terminal, two-thirds of the colicin E1 molecule, does not significantly influence its channel properties. The channel-forming activity of both polypeptides is dependent upon the presence of a membrane potential, negative on the trans side of the membrane. The average single channel conductance of colicin E1 and the COOH-terminal fragment is 20.9 +/- 3.9 and 19.1 +/- 2.9 picosiemens, respectively. The rate at which both proteins form conducting channels increases as the pH is lowered from 7 to 5. Both molecules require negatively charged lipids for activity to be expressed, exhibit the same ion selectivity, and rectify the current to the same extent. Both polypeptides associate irreversibly with the membrane in the absence of voltage, but subsequent formation of conducting channels requires a negative membrane potential.

Amino Acid Sequence↗
PubMed1982

On a domain structure of colicin E1. A COOH-terminal peptide fragment active in membrane depolarization.

A prolonged digestion of colicin E1 with dilute trypsin results in the generation of a trypsin-resistant peptide fragment of the colicin which is approximately one-third of the molecule. The amino acid composition of the fragment, Mr = 20,000, is more nonpolar in nature than the colicin, with the major change in composition being the reduction of the arginine content from 25 residues in the Mr = 56,000 colicin molecule to approximately 1 residue in the fragment. The NH2-terminal amino acid sequence of the tryptic fragment shows no similarity to that of colicin E1. Alignment of this sequence with the complete amino acid sequence of the colicin E1 molecule obtained by others, shows that the fragment occupies all, or almost all, of the COOH-terminal section of the molecule. The fragment behaved similarly to colicin E1, in being able to dissipate a potassium diffusion potential in unilamellar membrane vesicles made of dimyristoylphosphatidylcholine. The fragment was able to dissipate the diffusion potential above and below the temperature region (Tm = 23.5-24 degrees C) of the lipid phase transition in these vesicles, consistent with a channel-like function. The fragment did not show measurable binding to colicin receptor sites on the cell surface, but was much more efficient than colicin E1 in its ability to inhibit proline transport by inner membrane vesicles of Escherichia coli. These data imply that a membrane channel-forming function of the molecule is located in this 20,000 molecular weight region at the COOH-terminal end of the colicin molecule.

Amino Acid Sequence↗
PubMed1981

Differential scanning calorimetry of chloroplast membranes: identification of an endothermic transition associated with the water-splitting complex of photosystem II.

The structure of spinach thylakoid membranes has been investigated by sensitive differential scanning calorimetry. Six endotherms are observed between 20 and 85 degrees C, corresponding to order--disorder transitions of different structural domains within the thylakoid membrane. In a medium of relatively high ionic strength, endothermic transitions occur at 42, 54, 65, 72, 79, and 84 degrees C, with the 65 degrees C transition being particularly prominent. At a lower ionic strength, transitions are centered at 44, 61, 66, 70, 78, and 83 degrees C. The 42--44 degrees C endothermic transition (the A transition) can be correlated with the modification of three electron-transport components or properties associated with photosystem II: (i) release of manganese from the membrane, (ii) the loss of O2 evolution with water as a donor, and (iii) a decrease in the redox potential of the hydroquinone-reducible cytochrome b-559. Both the A transition and the ability to evolve O2 are irreversibly lost after heating to 49 degrees C and also after exposure to trypsin, suggesting the involvement of protein in this transition. The interpretation of these observations is that one effect of the A transition involves the thermal disruption of a protein component on the donor side of photosystem II.

Calorimetry, Differential Scanning↗
PubMed1980

Reversal by trypsin of the inhibition of active transport by colicin E1.

The time course for inhibition of proline transport and irreversible loss of cell viability after treatment with colicin E1 was measured as a function of temperature between 13 and 33 degrees C, using a thermostatted flow dialysis system. Complete inhibition of proline transport at 33 and 13 degrees C occurred in 0.5 min and 3 to 5 min, respectively, after addition of colicin E1 at an effective multiplicity of about 4. At these times, the fractional cell survival, assayed by dilution directly from the flow dialysis vessel into trypsin, ranged from 35 to 80%, with viability always greater than 50% at the lower incubation temperatures. Further studies were carried out at 15 degrees C. Complete inhibition of proline transport, which required 2 to 3 min, occurred much more rapidly at 15 degrees C than did the decay of trypsin rescue, which required 10 to 15 min to reach a survival level of 10 to 20%. The direct addition of trypsin to the flow dialysis vessel, after an addition of colicin E1 that caused complete inhibition of proline or glutamine transport, resulted in restoration of net transport. The restored level was typically about 40% of the control rate, and was very similar to the fractional cell viability measured after incubation in trypsin in the same vessel. It is concluded that trypsin can restore active transport to a significant fraction of a cell population in which transport has been initially inhibited by colicin E1.

Biological Transport, Active↗
PubMed1979

Cytochrome f function in photosynthetic electron transport.

The questions of whether the stoichiometry of the turnover of cytochrome f, and the time-course of its reduction subsequent to a light flash, are consistent with efficient function in noncyclic electron transport have been investigated. Measurements were made of the absorbance change at the 553-nm alpha-band maximum relative to a reference wavelength. In the dark cytochrome f is initially fully reduced, oxidized by a 0.3-s flash, and reduced again in the dark period after the flash. In the presence of gramicidin at 18 degrees C, the dark reduction was characterized by a half-time of 25-30 ms, stoichiometries of cytochrome f:chlorophyll and P700:chlorophyll of 1:670 and 1:640, respectively, and a short time delay. The time delay in the dark reduction of cytochrome f, which is expected for a component in an intermediate position in the chain, becomes more apparent in the presence of valinomycin and K+. Under these conditions the half-time for cytochrome f dark reduction is 130-150 ms, and the delay is approximately equal to 20 ms. The measured value for the activation energy of the dark reduction of cytochrome f (11 +/- 1 kcal/mol) is the same as that for noncyclic electron transport in steady-state light. A sigmoidal time-course for the reduction of cytochrome f has been calculated for a simple linear electron transport chain. The kinetics for reduction of cytochrome f predicted by the calculation, in the presence of valinomycin and K+, are in reasonably good agreement with the experimental data. There is an appreciable amount of data in the literature to document complex properties of cytochrome f after illumination with short flashes, and evidence for complexity in a light-minus-dark transition. The data presented here, obtained after a long flash that should establish steady-state conditions, either fulfill or are consistent with the basic criteria for efficient function of cytochrome f in noncyclic electron transport.

Chlorophyll↗
PubMed1979

Photooxidation of the high-potential iron-sulfur center in chloroplasts.

The high-potential iron-sulfur center associated with the g = 1.89-1.90 electron paramagnetic resonance signal in spinach chloroplasts is fully reduced in the dark. It is photooxidized in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and reduced after illumination with red actinic light in the absence of inhibitors. The light-induced redox changes of the high-potential iron-sulfur center are shown to correlate with those of plastocyanin detected at g = 2.05. Demonstration of light-induced redox changes in the high-potential iron-sulfur center provides support for a functional role of this component in the chloroplast electron transport chain.

Journal Article↗
PubMed1977

Kinetics of the photoreduction of cytochrome b-559 by photosystem II in chloroplasts.

The kinetics of the photoreduction of cytochrome b-559 and plastoquinone were measured using well-coupled spinach chloroplasts. High potential (i.e, hydroquinone reducible) cytochrome b-559 was oxidized with low intensity far-red light in the presence of N-methyl phenazonium methosulfate or after preillumination with high intensity light. Using long flashes of red light, the half-reduction time of cytochrome b-559 was found to be 100 +/- 10 ms, compared to 6-10 ms for the photoreduction of the plastoquinone pool. Light saturation of the photoreduction of cytochrome b-559 occurred at a light intensity less than one-third of the intensity necessary for the saturation of ferricyanide reduction under identical illumination conditions. The photoreduction of cytochrome b-559 was accelerated in the presence of dibromothymoquinone with a t 1/2 = 25-35 ms. The addition of uncouplers, which caused stimulatory effect on ferricyanide reduction under the same experimental conditions resulted in a decrease in the rate of cytochrome b-559 reduction. The relatively slow photoreduction rate of cytochrome b-559 compared to the plastoquinone pool implies that electrons can be transferred efficiently from Photosystem II to plastoquinone without the involvement of cytochrome b-559 as an intermediate. These results indicate that it is unlikely that high potential cytochrome b-559 functions as an obligatory redox component in the main electron transport chain joining the two photosystems.

Chloroplasts↗
PubMed1976

An evaluation of N-phenyl-1-naphthylamine as a probe of membrane energy state in Escherichia coli.

Colicin El and the uncoupler of oxidative phosphorylation, trifluoromethoxy-carbonylcyanidephenylhydrazone (FCCP), cause an increase in the fluorescence intensity of N-phenyl-1-naphthylamine bound to whole cells of Escherichia coli. It has been shown elsewhere that this fluorescence increase correlates well with de-energization. Addition of glucose causes a large cyanide-sensitive decrease of intensity, tentatively associated with energization, with the emission spectrum almost returning to the original trace with a peak at 417 nm. These data suggest that there may be a measurable competition between de-energization and energization of the cell membrane, and that the probe fluorescence intensity may be a general indicator of membrane energy level. The conclusions reached about cellular energy level from measurements of the probe fluorescence intensity correlate partly (a, b below, not c) with the energy level assayed physiologically through rates of active transport; (a) FCCP is found to be a poor inhibitor of proline transport if cells are first incubated with glucose, showing eutger cinpetition between the processes of energization and de-energization or an increase in the envelope permeability barrier to FCCP caused by glucose addition. (b) Cyanide blocks the fluorescence decrease caused by glucose and inhibits proline and serine transport, consistent with the decrease in probe fluorescence intensity indicating an increase in membrane energization. However, (c) it appears that the amplitude of the fluorescence intensity decrease caused by glucose addition in the presence of FCCP and colicin E1 greatly exaggerates the extent of real membrane energization. Glucose added after uncoupler can cause only a small increase, and after colicin, a negligible increase in the proline transport rate, indicating that the magnitude of the fluorescence intensity decrease after glucose addition is not a true measure of membrane energization, but rather seems to amplify this energization greatly. Glucose addition does not cause a decrease in fluorescence intensity in cells treated with EDTA to remove lipopolysaccharide and an apparent barrier to the probe. The rotational relaxation time of the probe in intact cells appears to correlate somewhat better with the cellular energy level than does intensity.

1-Naphthylamine↗