Richard A. Hudson

Transport of pyrimidine nucleosides across human erythrocyte membranes.

Abstract The transport of pyrimidine nucleosides in human erythrocytes has been shown to occur by a facilitated diffusion mechanism. Transport studies of these nucleosides are not complicated by metabolic conversions as in the case of the action of phosphorylases and deaminases on purine nucleosides. The transport carrier K m and v max were K m = 3.5, 1.7, 1.3 and 0.7 mM and v max = 16.5, 6.9, 4.7 and 10.0 mM/min for cytidine, cytosine-β-arabinoside, thymidine and uridine, respectively. The initial rates for nucleoside exit competition experiments are in good agreement with those calculated using the above K m s and v max s. Some preliminary evidence that the pyrimidine and purine nucleosides are transported by a common system is also presented.

Isolation and composition of the carotenoid-containing oil droplets from cone photoreceptors☆

A procedure for isolating the carotenoid-containing oil droplets of cone retinal photoreceptors of Gallus domesticus is described. The oil droplets, composed almost entirely of neutral lipids and carotenoids, have been separated into ten chromatographic components. Similar separations have been carried out on the total retinal neutral lipids for comparison. The neutral lipids represented 26.1% of the total retinal lipid. Cholesterol, cholesterol ester, mono-, di- and triacylglycerols represented 92.6% of the total neutral lipid. Each of these and other minor neutral lipid components were also present in the lipids extracted from the isolated oil droplets in correspondingly similar concentrations. However, the concentrations of carotenoids were greatly enriched in the neutral lipids of the oil droplets. Each of the major fatty acyl-containing neutral lipids from the chromatography of oil droplet lipids is greatly enriched in polyunsaturated fatty acids when compared with the corresponding component from the total neutral lipid chromatography. In the acylglycerols and free fatty acid fraction from the oil droplets, linoleic and arachidonic acid together represented 52-83% of the total polyunsaturated fatty acids present. The remainder was generally distributed about equally among six other acids. Except for the diacylglycerol fraction, linoleic acid was usually the most enriched acid in a specific oil droplet fraction when compared with any other polyunsaturated fatty acids. A similar pattern of polyunsaturated fatty acid enrichment observed in the fatty acids of the outer segment phospholipids relative to the corresponding total phospholipid fractions of this cone rich retina (Johnston, D. and Hudson, R.A. (1974) Biochim. Biophys. Acta 369, 269) suggest possible metabolic relationships between the oil droplet neutral lipids and the outer segment membrane phospholipids of the cone photoreceptors. A mechanism for the accumulation of the carotenoids in the oil droplets is also discussed.

A continuous-flow, rapid-mixing, photolabeling technique applied to the acetylcholine receptor

A continuous-flow technique is described in which a photoaffinity label, membrane rich in acetylcholine receptor, and various effectors are rapidly mixed, passed through a delay tube, through a tube in which they are irradiated, and are collected in a tube containing quencher. Delay times as short as 20 ms between mixing and photolysis are achievable. Because the flow is continuous, milliliter volumes of membrane can be labeled in a single run, which is convenient for the analysis of both the functional effects and sites of photolabeling. Using this technique, we have found that receptor in its transitory, active state, in which the channel is open, is more susceptible to photolabeling by the noncompetitive inhibitor analog [3H] quinacrine azide than is receptor in either its resting or desensitized states, in which the channel is closed. This technique should prove generally useful for the photolabeling of transient conformational states of macromolecules.

Oil droplet carotenoids of avian cones—I. Dietary exclusion: Models for biochemical and physiological studies

Abstract 1. 1. The feasibility of producing colonies of Japanese quail ( Coturnix coturnix japonica ) with carotenoid-free (“colorless”) retinal oil droplets has been demonstrated by dietary exclusion of carotenoids. 2. 2. Adult quail maintained on carotenoid-free rations undergo a gradual reduction in blood and liver carotenoid level; their “colorless” offspring possess normal growth and reproductive characteristics as well as unaltered visual cell morphology. 3. 3. Zeaxanthin is the major carotenoid found in quail blood. 4. 4. Hydrolysis of quail retinal extracts yields zeaxanthin and astacene. The latter is derived from astaxanthin and/or its esters.

An enzyme-linked immunosorbent assay for antibody against acetylcholine receptor.

An enzyme-linked immunosorbent assay is described for measurement of antibody against Torpedo acetylcholine receptor. As here developed, the assay is highly sensitive, reproducible, and requires small quantities of immunological reagents. Relative measurements of antibody concentration by this method are proportional to those determined by radioimmunoassay.

Phospholipids of the cone-rich chicken retina and its photoreceptor outer segment membranes

Abstract The lipid compositions of the cone-rich chicken retina and its outer segment membranes have been determined. The whole retina contained 25.1% lipid by weight, 26.0% of which were neutral lipids. Isolated outer segments contained 31.5% lipid by weight, 24.1% of which were neutral lipids. Total cholesterol represented 37.3 and 91.6% of the neutral lipid fractions from whole retina and isolated outer segments, respectively. The major phospholipids of the whole retina and outer segment membranes were phosphatidylcholine and phosphatidylethanolamine which represent, respectively, 41.8 and 33.5% of the total phospholipid of the whole retina and 44.9 and 34.9% of the phospholipids of the outer segment membranes. Phosphatidylserine was present as only 3.9 and 1.7% of the total phospholipids of whole retina and outer segment membranes, respectively. Sphingomyelin was present in relatively high concentration (9.1%) in whole retinal phospholipids and was enriched (11.0%) in the isolated outer segment membrane phospholipids. Palmitic (16:0), stearic (18:0), oleic (18:1), arachidonic (20:4) and docosahexanoic (22:6) acids account for most (80%) of the fatty acid of whole retinal phospholipids. Concentrations of arachidonic and docosahexanoic acid were highest in the serine and ethanolamine phosphatides from whole retina. There was little enrichment of these acids in any of the phospholipids of the outer segment membranes; instead, they were very high in linoleic acid.

Models of intermolecular interactions involving proton transfer in biopolymers

The results of spectrophotometric and conductimetric studies of the ionization of several phenol derivatives in the presence of amines in dioxane and aqueous dioxane solutions, were used to delineate the ranges of solvent polarity over which 1:1 proton transfer complexes are formed. Increasing polarity favors the formation of proton transfer complexes up to a point determined by the nature of the acid-base pair. Proton transfer complexes display a measurable stability over an appreciable range of solvent polarity varying from low (D < 11) to medium (11 < D < 45). Their formation involves a free energy change ranging from −2.0 to −7.1 kcal · mole−1 and a decrease in enthalpy ranging from 6.1 to 16.8 kcal · mole−1. The effects of proton transfer complex formation between side chain groups of biopolymers are subdivided into structural, chemical and kinetic, and the relevance of the results obtained from model studies to biopolymers is discussed. It is suggested that formation of proton transfer complexes can affect the direction of conformational change in macromolecules and play an important role in the formation of oligomeric structures. The numerical inferiority of proton transfer complexes in biopolymers, relative to hydrophobic and hydrogen bonding interactions should be partially offset by the greater contribution of the former to the total free energy and enthalpy of formation of specific biopolymer conformations.

On the interaction of cobra venom protein cardiotoxins with erythrocytes.

The principally active hemolytic toxin (cardiotoxin) previously purified from the venom of the Thailand cobra, Naja naja siamensis, was shown to produce spontaneous twitching, contractures and membrane depolarization in sartorius muscles from the frog, Rana pipiens. Spontaneous twitching, observed at concentrations greater than 0.1 uM was completely abolished by addition of tetrodotoxin and not affected by d-tubocurarine. Dose and time dependent membrane depolarization of muscle fibers was observed to occur within 10-30 min at 0.2 to 1.0 uM concentrations of the toxin. These observations, taken together with an amino acid analysis characteristic of previously described cobra venom cardiotoxins, characterized this hemolytic toxin as a cardiotoxin. In the absence of EDTA the initial velocities of erythrocyte hemolysis for this toxin showed a sigmoidal concentration dependence which became hyperbolic in the presence of EDTA. The largest increases in hemolysis rates on addition of 1 mM EDTA were observed at low toxin concentrations. In the presence of EDTA extracellular and membrane associated divalent cations are complexed, thus alleviating their competition with toxin for binding to the membrane, a key and apparently rate-determining initial step which leads to hemolysis. In the presence of EDTA hemolysis rates increased linearly at low toxin concentration and reached an extrapolated maximum value at toxin concentrations at which, given its molecular dimensions, there are just sufficient toxin molecules to cover the entire membrane surface area provided by the erythrocytes.

Phthalate ester hydrolases and phthalate ester toxicity in synchronously developing larvae of the brine shrimp (Artemia)

Abstract Di-n-butyl phthalate (DNBP) is toxic to synchronously developing larvae of the brine shrimp, Artemia . The LD50 for 24 h exposure is approximately 30 μM (8 ppm). DNBP is concentrated by larvae, and maximal uptake of DNBP precedes the onset of mortality. At the time of maximal uptake, most of the DNBP remains in the form of the diester. By the time of maximal mortality nearly all of the DNBP has been converted to the monoester, n-butanol and possibly other polar metabolities. n-Butanol and mono-n-butyl phthalate directly incubated with larvae were nontoxic when tested in concentrations at which DNBP was toxic. Soluble enzyme(s) extracted from the hatched larvae, but not from the dormant embryos, can convert DNBP to its monoester and n-butanol. The amount of enzyme activity increased with larval development in parallel with the kinetics of acute toxicity. The enzyme may be significant in the developmental program as well as in the mediation or moderation of the toxic effects of DNBP in Artemia larvae.

Hydrogen bonding interactions of p-nitrophenol

Abstract The thermodynamic parameters of the formation of the 1:1 hydrogen bonded complex of p-nitrophenol with dioxane, triethylamine and n-butylamine in cyclohexane, were determined by ultraviolet absorption spectroscopy: −ΔH = 7.03, 10.1, 9.21 kcal.mole−1, −ΔG = 2.41, 4.18, 4.44 kcaLmole−1 and −ΔS = 15.5, 19.8, 16.0 e.u., respectively. Virtually all of the p-nitrophenol is hydrogen bonded when the concentration of the proton acceptor reaches 0.1–0.5 M. At higher concentrations, additional bathochromic shifts are observed in the p-nitrophenol absorption spectrum. The solvation of the 1:1 p-nitrophenol complex in the concentrated dioxane and triethylamine solutions appears to follow 3:2 and 3:4 stoichiometries, respectively. The free energy, enthalpy and entropy of the dioxane solvation were determined to be 1.3 kcal.mole−1, −5.1 kcal.mole−1 and −21 e.u., respectively. In the case of n-butylamine, a new band appears at ~370 mμ, which continues to shift with increase in n-butylamine concentration, ending at 395 mμ in pure n-butylamine. This band was assigned to the absorption of the proton transfer complex NO2 ⋯H+-NH2Bu.