Guanosine 5′-triphosphate and guanosine 5′-[βγ-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase

1. GTP, but not p[NH]ppG (guanosine 5′-[βγ-imido]triphosphate), abolishes the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. 2. When either GTP or p[NH]ppG alone stimulate adenylate cyclase, the enzyme senses only those lipid-phase separations occurring in the inner half of the bilayer. 3. Trypsin treatment of intact hepatocytes has no effect on the basal, fluoride-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, ¹²⁵I-labelled-glucagon specific binding decays with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. 4. When GTP or p[NH]ppG are added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity is substantially increased and the decay plots are no longer first-order. 5. Trypsin treatment of purified rat liver plasma membranes abolishes basal and all ligand-stimulated adenylate cyclase activity, and ¹²⁵I-labelled-glucagon specific binding. 6. Benzyl alcohol activates the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities are affected differently when glucagon is present in the assays. 7. We suggest that guanine nucleotides alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.     

The reproductive behaviour ofAequidens vittatus (Pisces,Cichlidae) in Surinam,South America

Synopsis The behaviour of free-living pairs ofAequidens vittatus was observed in Surinam, South America. An ethogram of breeding behaviour is presented, based on those observations and on aquarium-held fish. This is a biparental, substrate-guarding species that spawns on a movable platform. Sexual differentiation of parental roles was more pronounced when the young were in the embryonic interval than when they were mobile juveniles. Females spent more time near their brood, attacked juvenile conspecifics more often, and fanned and mouthed embryos more than males did. Males were more involved than females in territorial spacing of pairs. The movable spawning leaf provides support for eggs on detritus substrate and may also provide protection against predators and rapid water level changes.     

Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.     

Macrobenthic community structure before and after pollution abatement in the Neches River estuary (Texas)

Macrobenthos and physicochemical conditions in the lower 39 km of the Neches River estuary were studied from August, 1984 to May, 1985. The results were compared with data collected in 1971–1972. Between 1972 and 1984 the permitted BOD waste load in the tidal Neches River was reduced from 123 125 kg d to 8717 kg d. River discharge and dissolved oxygen concentrations were consistently higher and salinity was lower, during the same seasons, during the 1984–1985 study. A total of 50 taxa of macrobenthos were collected in 1971–1972 and 104 taxa were collected in 1984–1985. The numbers of taxa per collection at each station in 1984–1985 were at least twice those found in 1971–1972. Minimum densities in 1984–1985 were much higher than the maximum densities in 1971–1972 at all stations. Patterns of dominance, Sorenson's similarity index, and diversity ( ) values indicated improved water quality in 1984–1985. Statistical analysis of macrobenthic diversity indicated significant differences between upper estuary and lower estuary stations in 1971–1972. No significant differences were found in 1984–1985. Significant differences in numbers of taxa, macrobenthos densities, and values between the two studies were found. Reductions of waste loads, increased river discharge, and deepening of the navigation channel were among the factors that probably contributed most to the changes in community structure of the macrobenthos observed.     

Evidence for two conformers of the beta subunit of tryptophan synthase in solution.

To explain our finding that the dimeric beta subunit of tryptophan synthase is only 50% inactivated by beta-chloro-L-alanine (Ahmed, S. A., Ruvinov, S. B., Kayastha, A. M., and Miles, E. W. (1991) J. Biol. Chem. 266, 21548-21557), we have extended our investigation using spectroscopic, steady-state kinetic, and electrophoretic methods. The spectroscopic properties of the half-active beta 2 dimer and the reactivation after alkali treatment show that the inactivation proceeds by an "enamine" mechanism. Although the fully active beta 2 dimer associates with the tryptophan synthase alpha subunit to form alpha 2 beta 2 complex, the inactive beta subunits in the half-active enzyme associate weakly or not at all with the alpha subunit. Our results provide evidence for two conformers of the beta subunit in solution: one is rapidly inactivated by beta-chloro-L-alanine and the other is not inactivated. Thermal inactivation studies and non-denaturing polyacrylamide gel electrophoresis of the half-active enzyme show that the beta 2 dimer exists in both homologous and heterologous combinations of these two forms. After removal of the reaction products and unreacted beta-chloro-L-alanine from the half-active beta 2 dimer by gel filtration, further incubation with beta-chloro-L-alanine results in the loss of 50% of the remaining activity. This result suggests that the subunits undergo rearrangement via an intermediate monomer form to regenerate the two conformers of the active beta subunit. This mechanism of rearrangement is supported by our finding that the extent of inactivation increases at lower concentrations of the beta 2 dimer.     

Hairpin formation in the self-complementary dodecamer d-GGTACGCGTACC and derivatives containing GA and IA mispairs

The dodecamer d-GGTACGCGTACC and four derivatives with GA and IA mispairs in the 6,7 and 5,8 positions have been examined in dilute solution and 0.01-0.1 M sodium chloride. Concentration dependence of Tm, gel electrophoresis, and equilibrium centrifugation indicate that these self-complementary oligomers can form hairpins under the present conditions. Thermal transitions measured in the ultraviolet primarily represent melting of hairpin to coil [cf. Scheffler et al. (1968, 1970)]. The Tm values show little or no depression for 6,7 substitution but rather large depression for 5,8 replacement. We interpret the results to indicate that the 6,7 sequences have two-base loops and five base pair stems and that the 5,8 sequences have four-base loops and four base pair stems. A concurrent theoretical modeling study [Raghunathan et al. (1991) Biochemistry (following paper in this issue)] provides support for this interpretation.     

Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor.

Plasminogen binding to cell surfaces results in enhanced plasminogen activation, localization of the proteolytic activity of plasmin on cell surfaces, and protection of plasmin from alpha 2-antiplasmin. We sought to characterize candidate plasminogen binding sites on nucleated cells, using the U937 monocytoid cell as a model, specifically focusing on the role of cell-surface proteins with appropriately placed lysine residues as candidate plasminogen receptors. Lysine derivatives with free alpha-carboxyl groups and peptides with carboxy-terminal lysyl residues were effective inhibitors of plasminogen binding to the cells. One of the peptides, representing the carboxy-terminal 19 amino acids of alpha 2-antiplasmin, was approximately 5-fold more effective than others with carboxy-terminal lysines. Thus, in addition to a carboxy-terminal lysyl residue, other structural features of the cell-surface proteins may influence their affinity for plasminogen. Affinity chromatography has been used to isolate candidate plasminogen receptors from U937 cells. A major protein of Mr 54,000 was recovered and identified as alpha-enolase by immunochemical and functional criteria. alpha-Enolase was present on the cell surface and was capable of binding plasminogen in ligand blotting analyses. Plasminogen binding activity of a molecular weight similar to alpha-enolase also was present in a variety of other cell types. Carboxypeptidase B treatment of alpha-enolase abolished its ability to bind plasminogen, consistent with the presence of a C-terminal lysyl residue. Thus, cell-surface proteins with carboxy-terminal lysyl residues appear to function as plasminogen binding sites, and alpha-enolase has been identified as a prominent representative of this class of receptors.