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.     

The determination of intracellular water space by NMR

A new method for the determination of intracellular water space using NMR spectroscopy is described. The method is based on the measurement of 59Co NMR signal intensity of an inert, stable and membrane-impermeable cobalt(III) compound such as Co(CN)3-6 or Co(imidazole)3+6 and the 2H or 1H NMR signal intensities of the freely permeable water. As an example of the method, the variation of the intracellular water space of human erythrocytes as a function of osmolality was measured.     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.     

Response of microbial populations to environmental disturbance

Taxonomic and genetic diversities of microbial communities disturbed by chemical pollutants were lower than in undisturbed reference communities. The dominant populations within the disturbed communities had enhanced physiological tolerances and substrate utilization capabilities, indicating that generalized physiological versatility is an adaptive characteristic of populations that successfully compete within disturbed communities.     

Production of carotene stereoisomers by Phycomyces blakesleeanus

Summary -Carotene steroisomers, mainly all-trans and to a small extent 9-cis, may be produced by the fungus Phycomyces blakesleeanus under normal fermentation conditions. The amount of the 9-cis--carotene may comprise up to 15% of the total -carotene. Similarly, cis-lycopene or-phytoene stereoisomers may be obtained when the fungus is fermented in the presence of specific -carotene inhibitors such as nicotine or diphenylamine respectively. This is the first report on the occurrence of cis-stereoisomers of carotenes in mycelial fungi.     

Measuring relative acetylcholine receptor agonist binding by selective proton nuclear magnetic resonance relaxation experiments.

A method is presented that uses selective proton Nuclear Magnetic Resonance (NMR) relaxation measurements of nicotine in the presence of the acetylcholine receptor to obtain relative binding constants for acetylcholine, carbamylcholine, and muscarine. For receptors from Torpedo californica the results show that (a) the binding constants are in the order acetylcholine greater than nicotine greater than carbamylcholine greater than muscarine; (b) selective NMR measurements provide a rapid and direct method for monitoring both the specific and nonspecific binding of agonists to these receptors and to the lipid; (c) alpha-bungarotoxin can be used to distinguish between specific and nonspecific binding to the receptor; (d) the receptor--substrate interaction causes a large change in the selective relaxation time of the agonists even at concentrations 100x greater than that of the receptor. This last observation means that these measurements provide a rapid method to monitor drug binding when only small amounts of receptor are available. Furthermore, the binding strategies presented here may be useful for the NMR determination of the conformation of the ligand in its bound state.     

Bacillus thuringiensis potency bioassays against Heliothis armigera, Earias insulana, and Spodoptera littoralis larvae based on standardized diets

Bacillus thuringiensis screening programs based on the official potency bioassay using third-instar larvae and on a neonate bioassay were developed for Heliothis armigera, Earias insulana, and Spondoptera littoralis. In these bioassays, the diets were standardized to be suitable, with minor modifications, for feeding of the three lepidopterans. The bioassay protocol was based on determination of the LC50 of the microbial standard HD-1-S-80 in the insects susceptible to B. thuringiensis var. kurstaki strains. This was followed by preliminary screening of B. thuringiensis strains at the LC50 of the B. thuringiensis standard. The B. thuringiensis strains causing 100% mortality at this LC50 in the larvae were selected for potency determinations. The neonate bioassay was suitable for accurate determinations of potencies also in S. littoralis--a representative of insects weakly susceptible to the HD-1 standard. The role of the official and the neonate bioassays in developing microbial control programs is discussed.     

Effect of Inhibitors on the Formation of Stereoisomers in the Biosynthesis of {beta}-Carotene in Dunaliella bardawil

An assay system was developed in which the effects of inhibitorsof ß-carotene biosynthesis in Dunaliella bardawilcould be tested. Since D. bardawil can be induced to accumulateover 10% of its dry weight as ß-carotene, it is particularlysuitable for such studies. Norflurazon a desaturation inhibitor,caused the accumulation of phytoene, or of phytoene and phytofluene,depending on the concentration employed. J-334, a substituted6-methylpyrimidine which also inhibits desaturation, causedthe accumulation of ß-zeacarotene, -carotene and phytoenein different proportions, depending on the concentration employed.The cyclization inhibitors, nicotine, CPTA and MPTA, severelyaffected the growth and survival of the alga, and their effectscould therefore not be studied directly. However, their actionwas observed indirectly by following the transformation of phytoenein norflurazon-pretreated phytoene-rich algae. Under these conditions,presence of the cyclase inhibitors caused the transformationof phytoene to lycopene, rather than to ß-carotene.The accumulated ß-carotene or the intermediates ß-zeacarotene,lycopene, -carotene, phytofluene and phytoene in D. bardawilwere all composed of two stereoisomers, tentatively assignedas the all-trans stereoisomer (55%) and the 9-cis stereoisomer(45%). This suggests that the isomerization reaction which leadsto the production of the presumed 9-cis isomers occurs earlyin the pathway of carotene biosynthesis, at or before phytoene,with no isomerization during the further transformations leadingto ß-carotene. (Received January 29, 1990; Accepted May 9, 1990)     

EFFECT OF LOW TEMPERATURE ON THE STEREOISOMER COMPOSITION OF β-CAROTENE IN THE HALOTOLERANT ALGA DUNALIELLA BARDAWIL (CHLOROPHYTA)1

Dunaliella bardawil Ben-Amotz & Avron, a β-carotene-accumulating halotolerant alga, was analyzed for the effect of growth temperatures on its pigment content and on the stereoisomeric composition of β-carotene by the use of advanced liquid chromatography and photodiode array detection. Decreasing culture temperature from 30° to 10°C increased the β-carotene content twofold and the ratio of 9-cis to all-trans β-carotene fourfold, with no significant changes in the other cell pigments. The variation of total β-carotene content by temperature was correlated with the integral irradiance received by the algal culture during a cell division cycle, whereas the 9-cis stereoisomer increased over the amount expected by that integration. The massive accumulation of 9-cisβ-carotene within the β-carotene globules is interpreted as indicating that the oily 9-cis stereoisomer protects against the crystallization of all-trans β-carotene at low temperatures.     

A Radioimmunoassay-Based Method for Measuring the True Affinity of a Monoclonal Antibody with Trace Amounts of Radioactive Antigen: Illustration with the Products of a Cell-Free Protein Synthesis System

This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen. It is based on a radioimmunoassay in which the antigen is labeled with radioactivity. It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling. It offers several advantages over previously described methods. Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen. It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex. It does not require the antigen or the antibody to be purified. In most cases, it requires no sophisticated equipment. This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.