Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla.

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules (SH (average sedimentation coefficient) = 12 400 S in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin (greater than or approximately 95% of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium. The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.     

Sucrose transport by Streptococcus mutans. Evidence for multiple transport systems

The transport of sucrose by selected mutant and wild-type cells of Streptococcus mutans was studied using washed cocci harvested at appropriate phases of growth, incubated in the presence of fluoride and appropriately labelled substrates. The rapid sucrose uptake observed cannot be ascribed to possible extracellular formation of hexoses from sucrose and their subsequent transport, formation of intracellular glycogen-like polysaccharide, or binding of sucrose or extracellular glucans to the cocci. Rather, there are at least three discrete transport systems for sucrose, two of which are $\text{phospho}\text{enol}\text{pyruvate-dependent}$ phosphotransferases with relatively low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values and the other a non-phosphotransferase (non-PTS) third transport system (termed TTS) with a relatively high apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. For strain 6715-13 mutant 33, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 6.25·10^?5 M, 2.4·10^?4 M, and 3.0·10^?3 M, respectively; for strain NCTC-10449, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 7.1·10^?5 M, 2.5·10^?4 M and 3.3·10^?3 M, respectively. The two lower $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ systems could not be demonstrated in mid-log phase glucose-adapted cocci, a condition known to repress sucrose-specific phosphotransferase activity, but under these conditions the highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ system persists. Also, a mutant devoid of sucrose-specific phosphotransferase activity fails to evidence the two high affinity (low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) systems, but still has the lowest affinity (highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) system. There was essentially no uptake at 4°C indicating these processes are energy dependent. The third transport system, whose nature is unknown, appears to function under conditions of sucrose abundance and rapid growth which are known to repress $\text{phospho}\text{enol}\text{pyruvate-dependent}$ sucrose-specific phosphotransferase activity in S. mutans. These multiple transport systems seem well-adapted to S. mutans which is faced with fluctuating supplies of sucrose in its natural habitat on the surfaces of teeth.     

Stereoselective preparation of deuterated reduced nicotinamide adenine nucleotides and substrates by enzymatic synthesis.

A-Side (4-R)-(4-²H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d₆ to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1-d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final $\text{A}{}_{260}\text{A}{}_{340}$ ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.     

Fractionation by velocity sedimentation of Torpedo nicotinic post-synaptic membranes

Velocity sedimentation on sucrose gradients containing Torpedo physiological saline has been utilized to fractionate Torpedo (Torpedo californica and T. nobiliana) post-synaptic membranes isolated initially on the basis of their density by equilibrium centrifugation. Membranes are separated into two populations: (1) those retained within the gradient (referred to as gradient pool); and (2) membranes sedimenting rapidly through the gradient (referred to as f 22, fraction 22 of the gradient). Comparison of their polypeptide compositions by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicates that the gradient pool consists of highly purified nicotinic post-synaptic membranes containing the peptides of the acetylcholine receptor and a peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000, while f 22 contains the contaminating membranes present in the initial suspension as well as a small fraction of the nicotinic post-synaptic membranes. On the basis of the kinetics of efflux of ²²Na⁺ from the membrane fractions, it is concluded that the gradient pool contains most of the sealed vesicles with functional nicotinic receptors. The internal volume (μl/mg protein) of those membranes exceeds that of f 22 by a factor of 4, and greater than 85% of that internal volume is equilibrated by the nicotinic agonist carbamylcholine, while for f 22 only 40% is equilibrated. Thin-section electron microscopy has been used to estimate the distribution of vesicle sizes. The observed distribution for the gradient pool indicates that these vesicles are a size homogeneous population of diameter 0.3 μm, while f 22 contains a number of smaller and larger vesicles. Torpedo post-synaptic membranes have been treated with alkali to remove the non-receptor peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000. After alkaline extraction, velocity sedimentation permits the isolation of a population of size-homogeneous and well-sealed vesicles containing only the peptides of the nicotinic receptor. It is concluded that upon homogenization, the innervated surface of the Torpedo electroplax tends to form vesicles of uniform size (0.3 μm) which can be readily isolated by velocity sedimentation and that the peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000 is not required for the maintenance of bilayer structure.     

A fluctuation analysis study of the development of amiloride-sensitive Na+ transport in the skin of larval bullfrogs (Rana catesbeiana)

In the presence of the Na⁺-channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX–XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value ($\text{S}{}_0$) decreased while the corner frequency ($\text{f}{}_{\mathrm{<mtext>c</mtext>}}$) increased as the mucosal amiloride concentration was increased from 0.5 to 24 μM. From the linear relationship between the $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ values and the amiloride concentrations it was possible to determine the binding ($\text{k′}{}_{01}$) and unbinding ($\text{k}{}_{10}$) constants for amiloride to its receptor on the Na⁺ channel. With these parameters as well as short-circuit current and $\text{S}{}_0$ values, the current through the individual Na⁺ channels ($\text{i}$) was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na⁺ channels ($\text{M}$) in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na⁺ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX–XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ with the amiloride concentration did not occur in these animals.     

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$, is resolved into a fast decaying or kinetic component, $\text{r}{}_{\mathrm{<mtext>f</mtext>}}$, and an infinitely slow decaying or static component, $\text{r}{}_{\mathrm{\infty }}$. The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; $\text{r}{}_{\mathrm{\infty }}$ is proportional to the square of the lipid order parameter. An empirical relation between $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{r}{}_{\mathrm{\infty }}$ is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.     

Glycolipids are not extracted from phospholipid bilayers by binding to ferritin-lectin conjugates

A radioactively-labelled glycosphingolipid, asialo-G_M1, has been incorporated into phosphatidylcholine multilamellar vesicles. After incubation with ferritin-Ricinus communis agglutinin 60 (RCA 60) conjugate at different temperatures, the vesicles were separated from the conjugate by discontinuous density gradient ultracentrifugation. Measurement of the distribution of the radioactively-labelled asialo-G_M1 in the pelleted conjugate fraction and freeze-etch electron microscopy of the vesicle fraction indicate that the decrease in labelling of vesicles by ferritin-RCA 60 conjugate with increasing temperatures (Tillack, T.W., Wong, M., Allietta, M. and Thompson, T.E. (1982) Biochim. Biophys. Acta 691, 261–273) reflects a decrease in apparent binding affinity rather than an ability of the conjugate to extract glycolipid from the phospholipid bilayer after binding.     

Single-electron transfer from NADH analogues to singlet oxygen

Laser flash photolysis techniques have yielded rate constants for physical and reactive quenching modes of $\text{O}{}_2\text{(}{}^1\text{Δ}{}_{\mathrm{<mtext>g</mtext>}}\text{)}$ by nicotine, nicotinamide adenine dinucleotide (oxidized and reduced forms) and the reduced forms of nicotinamide mononucleotide, nicotinamide adenine dinucleotide phosphate and nicotinamide hypoxanthine dinucleotide. In the case of the last four named compounds, kinetic spectroscopy furnished evidence for one-electron transfers to $\text{O}{}_2\text{(}{}^1\text{Δ}{}_{\mathrm{<mtext>g</mtext>}}\text{)}$. Specifically, production of O^?₂ was demonstrated unequivocally by reaction with 1,4-benzoquinone. Quantitative determinations revealed the extent of reactive quenching to be near 60% in each case.     

A chloride requirement for Na+-dependent amino-acid transport by brush border membrane vesicles isolated from the intestine of a mediterranean teleost (Boops salpa)

The uptake of d-glucose, 2-aminoisobutyric acid and glycine was studied with intestinal brush border membrane vesicles of a marine herbivorous fish: Boops salpa. The uptake of these three substances is stimulated by an Na⁺ electrochemical gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{C}{}_{\mathrm{<mtext>in</mtext>}}$). For glucose, an increase of the electrical membrane potential generated by a concentration gradient of the liposoluble anion, SCN^?, increases the Na⁺-dependent transport. This responsiveness to the membrane potential was confirmed by valinomycin. Differently from glucose, uptake of glycine and 2-aminoisobutyric acid requires, besides the Na⁺ gradient, the presence of Cl^? on the external side of the vesicles. In the absence of Cl^?, amino acid uptake is not stimulated by the Na⁺ gradient and is not influenced by an electrical membrane potential generated by SCN^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) or by a K⁺ diffusion potential ($\text{C}{}_{\mathrm{<mtext>in</mtext>}}\text{>C}{}_{\mathrm{<mtext>out</mtext>}}$). This Cl^? requirement differs from the Na⁺ requirement, since a Cl^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) does not result in an accumulation of glycine or 2-aminoisobutyric acid similar to that produced by an Na⁺ gradient.     

A stable traveling-wave solution of a model of cellular control processes with positive feedback

Edelstein's model

$\text{?E}\text{?τ}\text{=F(M, E)}$

,

$\text{?M}\text{?τ}\text{=G(M, E)+D}\text{?}{}^2\text{M}\text{?s}{}^2$

,

$\text{M(s,0)=?(s)}$

,

$\text{E(s,0)=ψ(s)}$

, where τ ? 0 and ?∞<s<∞, $\text{F(M, E>) = (K}{}_1\text{+M}{}^{\mathrm{m}}\text{)}\text{(K}{}_2\text{+M}{}^{\mathrm{m}}\text{)?k}{}_1\text{E, G(M, E)}\text{= k}{}_1\text{E ? k}{}_2\text{M,}$ m ? 2, describes the behavior of two basic chemical species during the cellular differentiation in a linear ensemble of the same cell type. We prove the existence and uniqueness of a travelling-wavefront solution. We also demonstrate one kind of stability for this solution.     

Inhibition of indolethylamine-N-methyltransferase by S-adenosylhomocysteine

$\text{S}$-Adenosylhomocysteine (SAH) is a potent product inhibitor for indoleamine-$\text{N}$-methyltransferase (INMT) from rabbit lung. The kinetic studies showed that this inhibition was competitive with respect to $\text{S}$-adenosylmethionine (SAM) and noncompetitive with respect to $\text{N}$-methylserotonin (NMS). The $\text{K}{}_{\mathrm{i}}$ value of 1.0 × 10^?5M indicated that SAH had a higher affinity than SAM or NMS for the enzyme. SAH seems to form a reversible complex with the enzyme.     

Altered DNA sedimentation by the presence of erythrocytes in alkaline sucrose gradient centrifugation

The presence of a relatively small number of red cells was found to affect DNA sedimentation profile of normal lymphocytes and acute leukemia cells, as observed by the alkaline sucrose gradient centrifugation technique coupled with the fluorometric measurement of DNA. Significant alteration was observed at a nucleated cell/erythrocyte ratio of $\text{20}\text{1}$ to $\text{0.2}\text{1}$, resulting in retardation of the $\text{S}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value and the entire sedimentation profile. This effect seemed to be rather specific to erythrocyte lysate, since corresponding amounts of erythrocyte ghost, IgM, bovine serum albumin, and an increased number of nucleated cells did not influence the profile to an appreciable degree.     

Influence of sterols on ion transport through lipid bilayer membranes

Charge-pulse relaxation experiments with the negatively charged lipophilic ions, dipicrylamine and tetraphenylborate, (as well as with the positively charged carrier system Rb⁺-valinomycin) have been carried out in order to study the influence of sterols on the ion transport through the lipid bilayer membrane. The mol fraction of the sterols (cholesterol, epicholesterol, ergosterol, stigmasterol, dihydrocholsterol, epicoprostanol and cholesterololeate) as referred to total lipid was varied in a wide range (mol fractions 0–0.8).The monoolein/sterol or dioleoylphosphatidylcholine/sterol mixtures were dissolved in $\text{n}$-hexadecane in order to minimize effects of the sterol on the membrane thickness.Cholesterol had a strong influence on the transport of the lipophilic ions. Its incorporation into monoolein membranes increased the rate constant ${}_{\mathrm{<mtext>i</mtext>}}$ of translocation up to 8-fold, but incorporation into phosphatidylcholine membranes had virtually no influence on $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$. The other sterols with one hydroxy group and cholesterololeate had no influence on the rate constant or the partition coefficient β. The results are discussed on the basis of a possible change of dipole potential of the membrane caused by cholesterol and its derivatives.In the case of valinomycin-mediated Rb⁺ transport only cholesterol had a strong influence on transport properties. The rate constants of association ($\text{k}{}_{\mathrm{<mtext>R</mtext>}}$) as well as the rate constants of translocation of the complex ($\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$) and of the free carrier ($\text{k}{}_{\mathrm{<mtext>S</mtext>}}$) were reduced by incorporation of cholesterol up to eight-fold. The decrease of $\text{k}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$ are possibly caused by a decrease of membrane fluidity, whereas the decrease of $\text{k}{}_{\mathrm{<mtext>R</mtext>}}$ may be due to an increase of surface potential. The different action of cholesterol on the two transport systems is discussed under the assumption that the adsorption plane of the lipophilic ion is located more towards the aqueous side and that of the ion-carrier complexes more towards the hydrocarbon side of the dipole layer.     

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (67 mM) and $\text{V}$ (30 μmoles · min^?1 ·g^?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with fructose 1.0 mM (0.7 mM), $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with ATP 0.54 mM (0.37 mM), $\text{V}$ 10.3 μmoles · min^?1 · g^?1 (10.1 μmoles · min^?1 · g^?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.