Oxidised glutathione reductase activity in mouse epidermis: TPA induced change and its modulation by vitamin A

Single cutaneous application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) increased epidermal oxidised glutathione reductase activity in adult mouse by almost 100%. Pretreatment of animals with vitamin A for a week resulted in 75% inhibition of TPA induced change in the enzyme activity which remained unaffected in skin treated with vitamin A alone. This biochemical change in skin induced by TPA and modulated by vitamin A has been discussed in relation to epidermal hyperplasia.     

Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s state III _s,a state II _s state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

Metabolism of D- and L-glyceraldehyde in adipose tissue: a stereochemical probe for glycerokinase activity

Distributions of (14)C have been determined in free glycerol, in glycerol from triglycerides, in glucose from glycogen, and in lactate after incubation of d-glyceraldehyde-3-(14)C and l-glyceraldehyde-3-(14)C with rat adipose tissue. The distributions are interpreted in terms of presently accepted possible reactions for the initial metabolism of glyceraldehyde. Formation of glycerol-1-(14)C from d-glyceraldehyde-3-(14)C indicates that in adipose tissue glyceraldehyde is reduced to glycerol. Incorporation of (14)C from d-glyceraldehyde-3-(14)C into carbon 3 of the glycerol of triglyceride indicates that d-glyceraldehyde is either phosphorylated or oxidized to d-glyceric acid, or both, in its initial metabolism. Incorporation of (14)C from l-glyceraldehyde-3-(14)C into carbon 3 of glycerol indicates that l-glyceraldehyde is reduced to glycerol, which is phosphorylated and (or) converted to d-glyceric acid via l-glyceric acid. Some (14)C from l-glyceraldehyde-3-(14)C is incorporated into carbon 1 of glycerol of triglycerides and carbon 4 of glycogen; the explanation for this incorporation is uncertain.     

Studies on the existence of a pathway in liver and muscle for the conversion of glucose into glycogen without glucose 6-phosphate as an intermediate

Mixtures of (14)C-labelled glucose plus pyruvate were incubated either with rat diaphragm or slices of rat liver. Incorporation of glucose carbon into glycogen was compared with its incorporation into glucose 6-phosphate relative to the incorporation of pyruvate carbon into these metabolic products. There was no preferential incorporation of glucose carbon relative to pyruvate carbon into glycogen compared with glucose 6-phosphate in the liver slices, but there was in diaphragm. On the assumption that glucose 6-phosphate is a necessary intermediate in the conversion of pyruvate carbon into glycogen, this is evidence for the existence in muscle, but not in liver, of more than one pool of glucose 6-phosphate or of a pathway from glucose to glycogen without glucose 6-phosphate as an intermediate. Galactose carbon, relative to pyruvate carbon, was preferentially incorporated into liver glycogen, so that a substrate converted in liver into glycogen without glucose 6-phosphate as an intermediate could be detected by this approach.     

Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.     

Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF)β-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGFβ-like activities at an apparent molecular weight of 6–10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal expiants. The relation between this activity and a recently described TGFβ-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.     

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     

Crystallization of alfalfa mosaic virus coat protein as a T = 1 aggregate

Reassembled alfalfa mosaic virus coat protein was partially digested with trypsin to remove the first 26 amino acids (Bol et al., 1974). These particles are empty icosahedral protein shells built with 60 alfalfa mosaic virus protein subunits. This aggregate has been crystallized in two different crystal forms, one of which diffracts X-rays to at least 3.4 Å resolution. The type I crystals (space group $\text{P6}{}_3\text{, a = 200}\text{A}\text{?}\text{, c = 314}\text{A}\text{?}$) contain two particles per cell separated by 195 Å with each sitting on a 3-fold axis. The type II crystals contain three particles per cell in space group P3₁or P3₂ ($\text{a = 201}\text{A}\text{?}\text{, c = 485}\text{A}\text{?}$). Other T = 1 viral particles have very similar diameters.     

The photosynthetic electron transfer chain of Chromatium vinosum chromatophores flash-induced cytochrome b reduction

Reduction of a cytochrome b following excitation by a single, short, near-saturating light flash has been demonstrated in Chromatium vinosum chromatophores. The extent of reduction is increased by addition of antimycin. The cytochrome has an α-band maximum at 562 nm in the presence of antimycin.The cytochrome b reduction is most readily observed in the presence of antimycin at high redox potential when cytochrome c-555 is oxidised before excitation. Under these conditions the half-time for reduction is about 20 ms, and the extent is about 0.5 mol of cytochrome b reduced per mol of reaction center oxidised. This extent of reduction is observed on the first flash-excitation from the dark-adapted state, and there was no indication that the reaction center quinone acceptor complex acted as a two-electron accumulating system. With cytochrome c-555 reduced before excitation, the extent of cytochrome b reduction is approximately halved. The factors which result in substoichiometric cytochrome b reduction are not yet understood.Agents which appear to inhibit primary acceptor oxidation by the secondary acceptor (UHDBT, PHDBT, DDAQQ, HOQNO, o-phenanthroline), inhibit reduction of the cytochrome b. DBMIB inhibits cytochrome b reduction but does not appear to inhibit primary acceptor oxidation.These observations confirm that a cytochrome b receives electrons delivered from the primary acceptor complex, and indicate that the photoreduced cytochrome b is reoxidised via an antimycin-sensitive pathway.