Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Photosynthetic pigment concentration, organization and interconversions in a pale green Syrian landrace of barley (Hordeum vulgare L., Tadmor) adapted to harsh climatic conditions

Tadmor is a Syrian barley landrace that has adapted to semi-arid environments. Its leaves are pale green because of a 30% decrease in the chlorophyll and the carotenoid content of the chloroplasts (leading to a 7·5% decrease in light absorption) compared with barley genotypes that are not adapted to harsh Mediterranean climatic conditions (e.g. Plaisant). This difference in pigment content was attenuated during growth of the plants in strong light, but was strongly amplified when strong light was combined with a high growth temperature. The low pigment content of Tadmor leaves was not associated with significant changes in the pigment distribution between the photosystems or between the reaction centres of the photosystems and their associated chlorophyll antennae. No significant difference in the photosynthetic activity (O₂ production per unit absorbed light) was observed between Tadmor and Plaisant. The conversion of violaxanthin to zeaxanthin in strong light and its reversal in darkness were much faster and operated at a higher capacity in Tadmor leaves compared with Plaisant leaves, resulting in an increased photostability of photosystem II in the former leaves. The accelerated xanthophylls interconversion in the Syrian landrace was associated with, and possibly related to, an increased fluidity of the thylakoid membranes. The lipid peroxide level was lower in Tadmor compared with Plaisant. In contrast, no difference was found in the non-photochemical quenching of chlorophyll fluorescence between the two barley genotypes. The data indicate that the pale green Syrian landrace is equipped to survive excessive irradiance through a passive reduction of the light absorptance of its leaves, which mitigates the heating effects of strong light, and through the active protection of its photochemical apparatus by a rapid xanthophyll cycling.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Liver fatty acid-binding protein in two cases of human lipid storage

Summary FABPs in the various tissues play an important role in the intracellular fatty acid transport and metabolism. Reye's syndrome (RS) and multisystemic lipid storage (MLS) are human disorders characterized by a disturbance of lipid metabolism of unknown etiology. We investigated for the first time L-FABP in these two conditions. Affinity purified antibodies against chicken L-FABP were raised in rabbits, and found to cross-react specifically with partially purified human L-FABP. L-FABP content in liver samples of two patients with RS and MLS was investigated by immuno-histochemistry, SDS-PAGE and ELISA. L-FABP immuno-histochemistry showed increased reactivity in the liver of RS patient and normal reactivity in MLS liver. L-FABP increase in RS liver was confirmed by densitometry of SDS-PAGE and ELISA method. By these two methods the increase amounted to 180% and 199% (p < 0.02), respectively, as compared to controls. A possible role of L-FABP in the pathogenesis of RS is discussed.     

Melanocortin‐1 receptor structure and functional regulation

The melanogenic actions of the melanocortins are mediated by the melanocortin‐1 receptor (MC1R). MC1R is a member of the G‐protein‐coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or α‐melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist‐independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.     

Competitive and facilitative relationships among three shrub species,and the role of browsing intensity and rooting depth in the Succulent Karoo,South Africa

Abstract. The nearest‐neighbour technique is used to infer competition and facilitation between the three most abundant species in a semi‐arid region of western South Africa. Relationships among the shrubs Leipoldtia schultzei and Ruschia robusta, which are leaf‐succulent members of the Mesembryanthemaceae (‘mesembs’) and Hirpicium alienatum a non‐succulent Asteraceae, were compared on two adjacent sites with different histories of browsing intensity. Competition was more prevalent and more important than facilitation. The only evidence for facilitation was found at the heavily‐browsed site where the palatable Hirpicium was larger under the unpalatable Leipoldtia. Generally the prevalence and importance of competition was reduced at the heavily‐browsed site. Strong evidence was obtained for intraspecific competition in each of the three species; also, competition was evident between the two mesembs, where Leipoldtia was competitively dominant over Ruschia, although neither species inhibited Hirpicium. Minimal competition between the mesembs and the asteraceous shrub was interpreted in terms of differentiation in rooting depth, and competition within the mesembs, in terms of overlap in rooting depth. The mesembs had the bulk of their roots in the top 5 cm of soil, while the asteraceous shrub had the bulk of its roots, and all its fine roots, at greater depths. The shallow‐rooted morphology of the mesembs is well adapted to utilize small rainfall events, which occur frequently in the Succulent Karoo, and do not penetrate the soil deeply. Modifications of existing methods are applied for analysing nearest‐neighbour interactions.     

An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.     

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Characterization of polymorphic microsatellite markers for the Carnaby's cockatoo (Calyptorhynchus latirostris) and related black cockatoo species

Microsatellite loci were isolated from Carnaby's black cockatoo (Calyptorhynchus latirostris: Aves), a highly valued, endangered, and endemic species of bird from Western Australia. This study describes three dinucleotide and one tetranucleotide microsatellite loci for which the primers produced clear and polymorphic amplification patterns with between two and nine alleles and moderate levels of variability. Two additional dinucleotide markers which were monomorphic in the Carnaby's cockatoo were able to amplify and were polymorphic in two other species of black cockatoo, greatly increasing the utility of these markers.     

Lipid changes during development of Blastocladiella emersonii

The lipid composition of swimming spores, cysts and five hour germlings was established. Spores utilized triglycerides first, then phospholipids. Upon encystment all glycolipid components decreased, while in germlings the phospholipids, monoglycerides and sterol esters exhibited a marked increase.