Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Enkephalin analogs and dermorphin in the conscious dog: Structure-activity relationships

Those structural features of enkephalins (ENK) responsible for in vitro organ bath and receptor binding activity have been investigated in detail in the conscious, chronically instrumented dog. Amide analogs of Leu⁵-ENK display reduced activity, which is restored by D-Ala² substitutions. N-terminal L-Tyr is required for full opiate activity. Although proven δ-receptor agonists do appear generally more active, distinctions made in vitro between μ and δ binding are not apparent in the complex hemodynamic responses which occur in the intact unanesthetized dog. The amphibian skin peptide dermorphin, which contains D-Ala², elevates heart rate, systemic arterial pressure, and induces vomiting with near maximal activity at a dose of 1.0 μg/kg; this activity is inhibited by naloxone. This activity, coupled with dermorphin's apparent presence in mammalian tissue, suggests that it may represent another peptide factor in cardiovascular regulation. In the conscious dog, ENK elevate heart rate and systemic arterial pressure; this activity does not appear to be fully explained by in vitro receptor models.     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic β-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in β-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba²⁺ was not inhibited by d-glucose. Ba²⁺ served as a substitute for Ca²⁺ both in maintaining the glucose metabolism after removal of extracellular Ca²⁺ and making it possible for glucose to stimulate insulin release. Furthermore, Ba²⁺ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the β-cell content of cyclic AMP. It is likely that the observed actions of Ba²⁺ are mediated by Ca²⁺, since Ca²⁺-dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba²⁺ specifically.     

The quantum yield for CO2 uptake in C3 and C4 grasses

The quantum yield for CO₂ uptake was measured in C₃ and C₄ monocot species from several different grassland habitats. When the quantum yield was measured in the presence of 21% O₂ and 340 cm³ m^-3 CO₂, values were very similar in C₃ monocots, C₃ dicots, and C₄ monocots (0.045–0.056 mole CO₂ · mole^-1 quanta absorbed). In the presence of 2% O₂ and 800 cm³ m^-3 CO₂, enhancements of the quantum yield values occurred for the C₃ plants (both monocots and dicots), but not for C₄ monocots. A dependence of the quantum yield on leaf temperature was observed in the C₃ grass, Agropyron smithii, but not in the C₄ grass, Bouteloua gracilis, in 21% O₂ and 340 cm³ m^-3 CO₂. At leaf temperatures between 22–25°C the quantum yield values were approximately equal in the two species.     

Hair morphology and the relationships of species inSolanum sect.Basarthrum

The usefulness of features of leaf hairs in distinguishing subgenera and sections is well documented in bothRhododendron andSolanum. In this analysis of the taxa ofSolanum sect.Basarthrum (23 species), and of a sample of closely related taxa from sect.Petota (22 species), it is shown that such features serve to delineate subsectional groups such as series and some species as well. SectionBasarthrum has an unexpected diversity of hair types. Although this group has been characterized by 2-celled bayonet hairs, more than one half of the taxa in the section bear multicellular finger hairs, and 3 species also possess branched hairs. Thus, major rearrangements of the species previously assigned to sect.Basarthrum are indicated or supported by pubescence features. The taxa studied from seriesEtuberosa andJuglandifolia (both of sect.Petota) show hair types that a) are relatively primitive for the section, and b) show linkage between sects.Petota andBasarthrum.     

ESR study of intercalation: quantitative evaluation of drug-DNA binding through competition with a spin-labeled 9-aminoacridine

Interaction of a spin-labeled 9-aminoacridine with DNA was studied by electron spin resonance spectroscopy. Accurate determination of the binding parameters, equilibrium dissociation constant (K_D), and total number of ligand-binding sites was obtained using Scatchard and Lineweaver-Burk plots. The competition between 9-aminoacridine and its spin-labeled derivative was examined by a similar analysis of the spin-label signals. The practical interest of this method lies in the fact that the precision and the simplicity of the measurements allow the quantitative determination of the binding capacity of any intercalative drug which interacts specifically with adenine/thymine bases of DNA.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

A fluorogenic method for the demonstration of human serum 5′-nucleotide phosphodiesterase isozymes after polyacrylamide gel electrophoresis

A rapid fluorogenic method for the demonstration of 5′-nucleotide phosphodiesterase in human serum has been developed. This method uses the substrate 4-methylumbelliferyl 5′-thymidylate impregnated in agarose gels or filter paper strips. Zymograms are developed in less than 30 min at 25°C, and the sensitivity of this method has been compared with that of the indigogenic method.     

On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b₅₀ and cytochrome b_?90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q^?_I) and the secondary acceptor ubiquinone (Q_II) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b₅₀ reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b₅₀ reduction increased about 10-fold as the E_h(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with E_m(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b₅₀ reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q^?_I and Q_II. It was an effective inhibitor of cytochrome b₅₀ photoreduction at E_h(7.0) 390 mV, but not at E_h(7.0) 100 mV. At the latter E_h, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q^?_I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E_h(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b₅₀ reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b₅₀ oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high E_h values. However, at lower redox potentials, cytochrome b₅₀ oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b_?90 in the rapid reactions of the cyclic electron transfer chain     

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg²⁺, and at pH 7.4 in the presence of Mg²⁺. Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.