On the glutamate transport through cell envelope vesicles of Halobacterium halobium

Glutamate uptake by envelope vesicles of Halobacterium halobium was measured. Previous authors showed that the glutamate uptake needs the illumination as well as Na+ gradient across the membrane. The latter is considered to be the driving force for the uptake. No satisfactory explanation for the necessity of the illumination has not been given. We found that in the absence of Cl- in the medium, only Na+ gradient was enough to induce the glutamate uptake, i.e. no illumination was needed. Glutamate uptake was measured with various strains of H. halobium. We found that the envelope vesicles prepared from strains containing no bacteriorhodopsin showed the glutamate uptake in the dark and in the presence of Cl- in the medium provided only that Na+ gradient is imposed.     

Determination of membrane potential with lipophilic cations: correction of probe binding

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined in the absence and presence of membrane potential. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–4) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the amounts of binding were proportional to the probe concentration in the medium when the concentration is dilute. Upon illumination, interior negative membrane potential is generated which induces the uptake of phosphonium cation probe. 2 μM were employed as the initial probe concentration. The real membrane potential was evaluated by means of extrapolation to the state of no binding: The values of for various probes are plotted against the binding coefficient. Here, C_i^app is the apparent intra-vesicular concentration of the probes which is calculated without consideration of bound probes. The ordinate intercept of the plot gives the true concentration ratio, and from this the membrane potential is evaluated. The membrane potential-dependent binding was analysed with a model: the membrane is split into two halves, outer and inner half, and the amounts of bound probes in each region are governed by the concentration in the contiguous solution. We obtained a formula which describes amounts of binding as a function of the membrane potential.     

A photosystem other than PS370 also mediates the negative phototaxis of Halobacterium halobium

Abstract It was found that a photorepellent system other than photosystem 370 (PS370) also controls the behavior of Halobacterium halobium . Both the dependence of background illumination and wavelength where the response showed maximum action distinguished that photosystem from PS370 whose photoreceptor pigment is thought to be an intermediate of s-rhodopsin (sR). A mutant strain that has no detectable activity in PS370 and in photoattractant response was isolated. This mutant strain showed the repellent response due to the new photosystem.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Sensitization of Edman amino acid derivatives using the fluorescent reagent, 4-aminofluorescein

The ability to analyze amino acid derivatives at the femtomole level is one of the most interesting challenges in the field of protein microsequencing. 2-Anilino-5-thiazolinone amino acids, obtained by Edman degradation, were quantitatively derivatized with fluorescent primary amines. The most fluorescent reagent tested was 4-aminofluorescein. The amino acid derivatives sensitized with this reagent were separated using reversed-phase high-performance liquid chromatography and identified at the 100 attomole level. Incorporation of this method into the operation of a conventional automated sequencer is also described.     

Evaluation of DAPI as a Fluorescent Probe for DNA in Viable Petunia Protoplasts

4',6-Diamidino-2-phenyl-indole (DAPI), is a fluorescent probe that specifically and quantitatively stains DNA. Electroporation of viable Petunia protoplasts in the presence of DAPI revealed integral fluorescence that was similar for both the electroporated and fixed protoplasts. indicating quantitative staining of DNA. DAPI fluorescence was localized in the nuclei of viable protoplasts of Petunia. Protoplasts had a short term viability of 56-65% of the control (non-electroporated. unstained) protoplasts as determined by fluorescein diacetate staining 24 hr following electroporation in the presence of DAPI. The majority (84% of the number originally cultured) of these protoplasts subjected to electroporation were able to form a cell wall, but most did not form microcalli because they were blocked in cell division. The three week plating efficiency for protoplasts exposed to DAPI was 4% of the original number of protoplasts initially cultured compared to 30% for the control. DAPI should not be used as a fluorescent probe for plant protoplasts when the protoplasts are cultured for sustained growth because the levels of DAPI required to obtain quantitative staining of the DNA resulted in inhibition of the cell cycle. DAPI may, however, be used as a fluorescent DNA probe for short term (24 hr) studies.     

Effect of salt on photocycle and ion-pumping of halorhodopsin and third rhodopsinlike pigment of Halobacterium halobium

The cytoplasmic membranes of Halobacterium halobium contain at least three retinal pigments: bacteriorhodopsin (bR), halorhodopsin (hR), and a third rhodopsinlike pigment (tR). The amplitudes of the phototransient in the photolysis of hR and tR were measured in various salt solutions. Halogen ion (except fluoride) was required to retain the photocycle of hR. Parallels between the amplitude of the phototransient of hR and the magnitude of the photo-induced tetraphenylphosphonium (TPP+) uptake suggests that hR is a light-driven halogen pump, which supports the hypothesis by Schobert and Lanyi (J. Biol. Chem., 1982, 257:10306-10313). The order of effectiveness of halogen was Br- greater than Cl- greater than I-. On the other hand, no specific ion was required to retain the photocycle of tR, and tR was concluded to be nonelectrogenic.     

Fluorometric estimation of surface potential change associated with chemotactic stimulation in Tetrahymena pyriformis

For the quantitative estimation of surface potential change in intact cells a method was devised with the use of fluorescent probes, 8-anilino-1-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPN). Estimated values in liposomes were compared with changes in the zeta potential determined from electrophoresis. Both values agreed within the experimental variation, showing the usefulness of the method. The method was also applied to Tetrahymena pyriformis, which exhibits chemotaxis to various chemical stimuli. The surface potential change was observed when the cell was stimulated not only by inorganic salts but also by electrically neutral, hydrophobic compounds. The surface potential started to change in accordance with the depolarization of the membrane potential, except for the case of K⁺. Changes in the surface potential of T. pyriformis in response to Ca²⁺ and K⁺ were compared with those in the membrane potential. The quantitative contribution of the surface potential to cell depolarization associated with chemoreception is discussed.     

Gustatory responses of eel palatine receptors to amino acids and carboxylic acids

The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.     

Conformational change accompanying formation of oligomycin-induced Na(+)-bound forms and their conversion to ADP-sensitive phosphoenzymes in Na+,K(+)-ATPase.

Oligomycin reduced the fluorescence intensity of an N-(p-(2-benzimidazoly)phenyl) maleimide (BIPM) probe at Cys-964 of the alpha-chain of pig kidney Na+,K(+)-ATPase with increase in the concentration of Na+ with a Hill coefficient of nh = 0.77 with Kh = 231 mM. The maximum fluorescence decrease was around 80% of the value observed after accumulation of ADP-sensitive phosphoenzyme (E1P) in the presence of 2 M Na+. The addition of Mg2+ and ATP with Na+ or choline chloride to give the same final ligand concentration to the Na(+)-enzyme-oligomycin complex formed with 16 mM Na+ + 1,984 mM choline chloride or 2 M Na+ induced rapid phosphorylation (20 or 21/s) and slower fluorescence decrease (12.1 +/- 1.2 or 10.1 +/- 3.2/s). These additions to the Na(+)-enzyme complex formed under the former or the latter conditions induced slow phosphorylation (13/s) prior to a much slower fluorescence decrease (3.4 +/- 0.3 or 8.6 +/- 0.7/s). The addition of Ca2+ and ATP to these enzyme complexes induced rapid fluorescence changes (21-11/s) followed by one order of magnitude slower rates of phosphorylation (1.5-1.3 s). These data suggest that the decrease in BIPM fluorescence induced by ATP with Ca2+ or with Mg2+, reflects the change of the Na+ binding state before or after the formation of E1P, respectively.