Characterization of transducin from bovine retinal rod outer segments. Participation of the amino-terminal region of T alpha in subunit interaction

The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.     

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.     

Determining the kinetics of membrane pores from patch clamp data without measuring the open and closed times

We present a new and very time saving way to determine kinetic rate constants from patch clamp data by using the correlation functions utilized in analyzing experiments with photon correlations.     

Disruption of energy metabolism and smoltification during exposure of juvenile atlantic salmon (Salmo salar) to low pH

Juvenile Atlantic salmon were held for 76 days at pH 4.7 during the period when the final stages of smoltification normally occur. Control salmon (pH 6.5) had significant increases in weight, length and liver somatic index which were not observed in those held at low pH. Condition factor decreased in both groups but to a significantly greater extent in the low pH group. After 15 days levels of ADP and glucose were higher and AEC, CP and glycogen were lower in muscles of salmon exposed to low pH. These qualitative differences were maintained until the end of the experiment. ATP and total adenylate concentrations in muscle were lower after 62 days of exposure to low pH compared to controls. The levels of ATP, total adenylates, AEC and glucose were consistently higher in livers of salmon exposed to low pH than those of controls. There were no differences in liver concentrations of AMP, CP and glycogen between the two treatments or with time of exposure within each treatment. The results suggest that exposure of juvenile Atlantic salmon to low pH increased gluconeogenesis and decreased food intake to the detriment of growth.     

Identification and purification of overlapping cosmid clones of the region Xq24-qter of human X chromosome using HPLC.

The assembly of a large physical map of genomes requires simultaneous analysis of many cosmid clones for overlapping regions. The search for overlapping regions may be achieved by various means. High-performance liquid chromatography (HPLC) provides an alternative to gel electrophoresis since microgram amounts of each DNA fragment may be collected into individual test tubes for further analysis. HPLC has been used to identify overlapping cosmid clones from a pool of cosmid DNA containing the terminal portion of the long arm of the human X chromosome (Xq24-qter). Among 400 cosmids analyzed, 3 were shown to overlap.     

Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins

The Escherichia coli RecB protein, normally synthesized in low amounts, has been amplified by linkage of the recB gene to the phage lambda leftward promoter in an expression plasmid. From strains harboring this plasmid, RecB protein has been purified to homogeneity by a simple procedure which includes affinity chromatography on a column of RecC protein bound to agarose. The purified RecB protein has DNA-dependent ATPase activity but no exonuclease activity. RecC protein alone has neither ATPase nor exonuclease activity. However, when combined together, the RecB and RecC proteins show the ATP-dependent double-stranded exonuclease properties characteristic of the RecBC DNase.     

Separation of Legionella pneumophila proteases and purification of a protease which produces lesions like those of Legionnaires' disease in guinea pig lung

Six discrete protease activities were recovered from the supernatant broth of Legionella pneumophila cultures by ion-exchange chromatography. One of these demonstrated in vitro activity against collagen, casein and gelatin. When administered into the lungs of guinea-pigs this protease elicited lesions which were pathologically similar to those seen in clinical and experimentally induced Legionnaires' disease.     

Xyloside transport by XylP, a member of the galactoside-pentoside-hexuronide family.

This paper describes the functional characterization of the xyloside transporter, XylP, of Lactobacillus pentosus with the aid of a spectroscopy-based assay system. In order to monitor the transport reaction, the natural xyloside isoprimeverose, a building block of hemicellulose, and the analogue methyl-isoprimeverose were chemically synthesized by a new and efficient procedure. The XylP protein was purified by metal affinity chromatography, following high level expression in Lactococcus lactis from the nisin-inducible promoter. The purified XylP protein was incorporated into liposomes, in which the glucose dehydrogenase from Acinetobacter calcoaceticus (sGDH) was entrapped. sGDH can oxidize aldose sugars in the presence of dichlorophenol-indophenol as electron acceptor. The coupled assay thus involves XylP-mediated isoprimeverose uptake followed by internal oxidation of the sugar by sGDH, which can be monitored from the reduction of 2,6-dichlorophenol-indophenol at 600 nm. The uptake of isoprimeverose was stimulated by the presence of the non-oxidizable methyl-isoprimeverose on the trans-side of the membrane, indicating that exchange transport is faster than unidirectional downhill uptake. Unlike other members of the galactoside-pentoside-hexuronide family, XylP does not transport monosaccharides (xylose) but requires a glycosidic linkage at the anomeric carbon position. Consistent with a proton motive force-driven mechanism, the uptake was stimulated by a membrane potential (inside negative relative to outside) and inhibited by a pH gradient (inside acidic relative to outside). The advantages of the here-described transport assay for studies of carbohydrate transport are discussed.