Richard W. Hendler

Procedure for simultaneous assay of two β-emitting isotopes with the liquid scintillation counting technique

Two liquid scintillation counting techniques are described for the simultaneous assay of two BETA -emitting isotopes. Both methods are based on the empirical determination of the energy distribution for each isotope in various combinations of windows and the effects of known amounts of quenching on these distributions. One method is applicable for any level of radioactivity from extremely low to high ranges while the second is applicable for high levels of activity. Applications of the methods for the isotope pairs C/sup 14/, H/sup 3/ and C/sup 14/, P/sup 32/ are discussed. (C.H.)

Comparison of energy-transducing capabilities of the two- and three-subunit cytochromes aa3 from Paracoccus denitrificans and the 13-subunit beef heart enzyme

In the accompanying paper, we have shown that the two-subunit cytochrome aa3 isolated from Paracoccus denitrificans displays the same kind of complex and interactive redox behavior as the 13-subunit cytochrome aa3 from beef heart. Therefore, the redox characteristics are not dependent on the additional 11 subunits. In the current work, we have examined the energy-transducing capabilities of both the two- and three-subunit enzymes obtained from Paracoccus denitrificans in relation to that of the 13-unit mammalian enzyme. We have found that in all of the tested functions, which included the development of delta psi and delta pH, and the pumping of protons, that the two-subunit enzyme is at least as efficient as the structurally more complex mammalian enzyme. There is thus a correlation between the complex redox behavior and energy transducing capabilities of the two enzymes. There was also no difference in energy-transducing capabilities between the two- and three-subunit forms of the bacterial enzyme. It seems that only 2 subunits are required for an efficient energy-transducing cytochrome aa3. The most likely role of the additional subunits in the mammalian enzyme, therefore, seems to be in regulation.

Magainin 2 amide and analogues Antimicrobial activity, membrane depolarization and susceptibility to proteolysis

We compared the abilities of synthetic magainin 2 amide and its analogues to inhibit the growth of Escherichia coli and to cause membrane depolarization in E. coli cells and cytochrome oxidase liposomes. The analogue, magainin A, was about 40‐times more active than magainin 2 amide in inhibiting the growth of E. coli and had a much more sustained effect on the membrane potential. In the liposomal system, however, there was only approx. 20% difference between these two peptides in the reduction of membrane potential and uncoupling of respiration. Studies with pronase digestion suggested that the difference in potency may be due to differential susceptibility to proteolysis in the presence of membranes.

Interactions between a new class of eukaryotic antimicrobial agents and isolated rat liver mitochondria.

Members of a newly discovered class of eukaryotic antimicrobial peptides are shown to release respiratory control in isolated rat-liver mitochondria. They also dissipate the membrane potential and inhibit respiration. The uncoupling activity of the peptides decreases with time probably due to the presence of proteases in the mitochondrial preparation. Quinine and Mg2+ reduce the activity of the peptides. The nature of the dependence of the respiratory rate on the concentration of added peptides suggests that they are active in a multimeric form, consistent with the formation of a channel across the inner mitochondrial membrane. The channel allows passage of sucrose.

Self-Absorption Correction for Carbon-14 A new treatment yields a correction factor that is linearly related to the thickness of the sample

A new and simple technique has been developed (10) for correcting C14 radioactivity measurements for loss of radiation due to self-absorption. Results are discussed which show the applicability of the technique for many different counting situations. It is found that the absorption of radiation follows a hyperbolic law much more closely than an exponential one. Furthermore, the absorption coefficient for the sample itself has been found to be a function of weight rather than a constant, as had been assumed for the derivation of the law of exponential absorption.

CHEMICAL AND FUNCTIONAL STUDIES ON THE IMPORTANCE OF PURPLE MEMBRANE LIPIDS IN BACTERIORHODOPSIN PHOTOCYCLE BEHAVIOR

In native purple membrane (PM), there are approximately 1 squalene, 2 glycolipid sulfate (GLS), and 6 phospholipid (PL) molecules per bacteriorhodopsin (BR) monomer [10]. Brief (∼ 2 min) exposure to 0.1% Triton X‐100 removes about 25%, 20%, and 6% of squalenes, GLS, and PL, respectively (this paper) while causing profound changes in the BR photocycle, including the loss of ‘photocooperativity’ [1]. The BR photocycle in Triton‐treated PM can be restored to near normal behavior by reconstitution with native PM lipids. Isolated squalenes are not effective whereas PL alone partially restores normal photocycle characteristics.

Some properties and the possible metabolic significance of amino acid-lipid complexes

Abstract After incubation of hen oviduct tissue with radioactive amino acids under various conditions, the lipid fraction was extracted from the tissue. It was found that the amino acids were bound to components in the lipif fraction and that the resulting complexes could be separated into discrete fractions by countercurrent distribution and column chromatography. The phenomenon was general for the eleven amino acids tested. The formation of the most nonpolar radioactive complex was inhibited by processes which inhibited protein synthesis. The results are discussed in terms of the possible involvement of these complexes in the metabolism of the amino acid.

Redox properties of b-type cytochromes in Escherichia coli and rat liver mitochondria and techniques for their analysis

We describe here apparatus and procedures for conducting potentiometric titrations and for analyzing the collected data in terms of the number of components present, their amounts and their midpoint potentials. Using these procedures we have determined the presence of three forms of cytochrome b1 in Escherichia coli with midpoint potentials at pH 7.1 OF about minus 50, plus 110 and plus 220 mV. We were not able to demonstrate a change in any of these potentials by the addition of phosphate, ATP, or 2, 4-dinitrophenol. We have been able to confirm the presence of two forms of cytochrome b in non-energized mitochondria and the apparent conversion of the low-potential component to a new high potential component upon energization of the mitochondria. However we cite further experimental data that question the actual conversion of one form of cytochrome b to another. An alternative interpretation based on our analysis suggests that the high voltage component may be present in a masked form in the non-energized mitochondria.

Some pitfalls in curve-fitting and how to avoid them: a case in point.

When curve-fitting is used to support a complex nonlinear model containing several exponential terms, some of which have closely-spaced time constants, a particular burden of proof must be assumed. Most important, the uniqueness of the solution must be explored and discussed. Statistical tests for the degree of error and independence of the parameters should be provided, as well as information relating to the steps actually used in the fitting procedures. As an example of the need for the procedures we recommend in this communication, we have chosen an important case in point that has been published recently, and which deals with the kinetics of electron transfer from fully-reduced cytochrome oxidase to O2, analyzed by the method of SVD-based least squares. The problems we deal with in this case are applicable to a wide variety of other cases that involve curve-fitting to mathematical models.

On the cytological unit for protein synthesis in vivo in E. coli: II. Studies with intact cells of type B

Abstract Intact cells of Escherichia coli B were pulse labelled with [ 14 C]amino acids, disrupted by shaking with glass beads and fractionated into membrane, ribosome, and soluble fractions. An additional ribosome fraction was obtained from the membranes by treatment with deoxycholate. It was found that although the ribosomes from both sources showed the same kinetics for uptake and loss of radioactivity, the specific radioactivity of the ribosomes obtained from the membrane was consistently higher than that obtained in the free ribosome fraction. The findings were considered in terms of a membrane-linked ribosome as an important functional unit for protein synthesis in Escherichia coli .