Interaction of epidermal growth factor with micelles monitored by photochemically induced dynamic nuclear polarization-1H NMR spectroscopy

Photochemically induced dynamic nuclear polarization (CIDNP)-1H-NMR spectroscopy has been used to study the interaction of the protein hormone epidermal growth factor (EGF) with micelles of sodium dodecyl sulfate (SDS) and dodecylphosphorylcholine (DPC). Conventional 1H-NMR spectra show that most protein resonances remain unperturbed when micelles are added to solution, which argues that the overall protein conformation is maintained in the presence of SDS or DPC at the concentrations used. Photo-CIDNP enhancements of resonances assigned to aromatic side chains of residues at the COOH terminus and beta-sheet regions of murine EGF (i.e. Trp-49, Trp-50, and Tyr-37) are considerably reduced in the presence of micelles, while resonances of aromatic side chains of residues found elsewhere on the protein surface are mostly unaffected. This suggests that the primary interaction between murine EGF and the micelle occurs at the micelle-bulk solvent interface. The overall negatively charged surface of SDS micelles tends to induce a stronger interaction with the protein compared to the zwitterionic DPC micelles, probably due to electrostatic interactions. Cleavage of the COOH-terminal pentapeptide containing both tryptophan residues enhances the already present, but weak, interaction with Tyr-10 and attenuates it with Tyr-37. A similar interaction pattern is found with rat EGF suggesting that at least concerning these two species of EGF the interaction is somewhat specific and conserved. A simple mass-action model for protein-micelle interaction is also presented.     

Heparan sulfate proteoglycans of human lung fibroblasts. Structural heterogeneity of the core proteins of the hydrophobic cell-associated forms

Heparan sulfate proteoglycans (HSPG) were solubilized from human lung fibroblast monolayers with detergent. Presumptive membrane-associated forms displaying hydrophobic properties were purified by gel filtration on Sepharose CL-4B, by ion-exchange chromatography on Mono Q and by incorporation in lipid vesicles. The HSPG preparations were 125I-iodinated and treated with heparitinase before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five radiolabeled proteins with apparent molecular weights of 125,000, 90,000, 64,000, 48,000, and 35,000 were visualized by autoradiography. A sixth protein, identified in nonreduced 125I-HSPG preparations, appeared as a non-HS chain-bearing Mr 35,000 peptide which was disulfide-linked to an HS chain-bearing peptide of similar size. This multiplicity of core proteins did not seem to result from proteolysis during the heparitinase treatment itself, since some of the core proteins migrated independently during gel filtration before heparitinase digestion. Moreover, heparitinase digestion of 125I-HSPG purified by affinity chromatography on an immobilized monoclonal antibody yielded only the Mr 64,000 protein. Alternative depolymerizations of the HS chains by heparinase or HNO2 also yielded multiple protein bands. These results imply that heterogeneity of the core protein moiety may be a genuine property of the hydrophobic HSPG of human lung fibroblasts. The occurrence of multiple integral membrane HSPG forms may be relevant for the multiple functions that have been ascribed to cell-surface HSPG.     

Complementarity of particles and pits in freeze-fractured hepatic and cardiac gap junctions

Summary Particles and pits of freeze-fractured gap junctions are considered as complementary structures despite the frequent observations of more regular and closer spacings of pits, ascribed to plastic deformation of particle arrays. Recently, however, the noncomplementarity of pits and particles in Purkinje fibers has been reported. To ascertain the relationship between both structures, gap junctions from fixed, cryoprotected liver and myocardium were investigated using spacing and density measurements and complementary replicas.In hepatocyte gap junctions, the center-to-center distances (mean±sd) among pits, 9.57±1.49 nm, and particles, 9.70±1.77 nm, are not significantly different. Density determinations yielded a slightly higher value for the pits, (11,510±830)/m², than for the particles, (11,230±950)/m². In the myocardium, the spacing of the regularly arrayed pits, 9.55±1.33 nm barely exceeds the value of 9.44±1.62 nm for the particles, which show some clustering. However, the packing density for the pits, (10,090±740)/m², appears a little higher than that of the particles (9,890±920)/m². As density and spacing measurements provided no decisive answers, the positions of individual pits and particles of complementary junctional faces were recorded on transparent sheets and compared. In this fashion, a one-to-one correspondence between particles and pits could be established, while small discrepancies may be attributed to plastic deformation. Moreover, the collinearity of pits and particles may be suggested by the observation of a platinum grain in the center of many pits.     

Characterization of murine monoclonal antibodies to human interferon-gamma (IFN-gamma) and their application for sandwich enzyme-linked immunosorbent assay (ELISA)

The three murine monoclonal antibodies (MAb), D1G2, D9D10, and D13C8, are specific for human interferon-gamma (IFN-gamma), but not human IFN-alpha and IFN-beta. They react weakly with heat-treated IFN-gamma. The three antibodies recognize different epitopes of the IFN-gamma molecule, as evaluated by antibody-binding inhibition experiments. We have used these three monoclonal antibodies to construct a sandwich enzyme-linked immunosorbent assay (ELISA). The best result was obtained when we used D1G2 or D9D10 MAb as a solid-phase immunosorbent and D1G2 or D9D10 MAb as a tracer. When we measured IFN-gamma in sera by a combination of D1G2 (a solid-phase) and D1G2 (a tracer), a result similar to the one by a combination of D9D10 (a solid-phase) and D1G2 (a tracer), was obtained. This may suggest that human IFN-gamma exists in oligomeric form. Recombinant human IFN-gamma expressed in E. coli is detectable at a concentration of 1 ng/ml in this sandwich ELISA. This assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule as well as measurement of IFN-gamma in human sera and tissue culture fluids.     

Dynamics of long-leaved sundew Drosera intermedia populations at two extremes of a hydrological gradient

Densities of Drosera intermedia were low in two studied habitats (10–25 ramets m⁻¹), a path through a wet heath (short inundation in spring, low soil moisture in summer) and a pool edge (longlasting inundation, high soil moisture in summer). The low densities could be explained by the observed low recruitment and high adult mortality.
The low recruitment resulted from: (1) a high first year mortality of the large number of seedlings that emerged each year in the path population, caused by summer drought and cover with algae after heavy rainfall; (2) the absence in two years out of three of seedling emergence at the pool edge, due to the longlasting inundation. In neither population any seedlings survived to flower; (3) low vegetative reproduction rate.
Adult mortality during the growing season was caused by drought, which did not occur at the pool edge. Rapid senescence in autumn, caused by summer drought on the path and by a rapid submersion after heavy rainfall at the pool edge, was associated with a high winter mortality.     

Differentiation between Xanthomonas campestris pv. graminis (ISPP List 1980), pv. phleipratensis (ISPP List 1980) emend., pv. poae Egli and Schmidt 1982 and pv. arrhenatheri Egli and Schmidt 1982, by Numerical Analysis of Phenotypic Features and Protein Gel Electrophoregrams

A numerical analysis of 257 phenotypic features of 45 bacterial isolates from grasses, revealed three phenons corresponding to (i) X. campestris pv. graminis (ISPP List 1980), (ii) X. campestris pv. phleipratensis (ISPP List 1980) and (iii) X. campestris pv. poae Egli and Schmidt 1982 and X. campestris pv. arrhenatheri Egli and Schmidt 1982. In each phenon, the strains clustered together regardless of the geographical origin of the isolates orthe year of isolation. Polyacrylamide gel electrophoresis of soluble proteins and host range studies, revealed four groups corresponding to the pathovars mentioned above. The four pathovars constitute definite biological entities that can be differentiated by phenotypic, gel electrophoretic and host range features.     

Steroid sulfatase activity in a Peptococcus niger strain from the human intestinal microflora.

A strictly anaerobic gram-positive coccus, identified as Peptococcus niger, that developed sulfatase activity towards steroid-3-sulfate esters was isolated from human fecal material. This strain desulfated the arylsulfate esters estrone-3-sulfate (100%) and beta-estradiol-3-sulfate (50%); only trace amounts of desulfated estriol-3-sulfate were found. In addition, alkylsulfatase activity was found towards the 3 alpha-sulfates of 5 alpha-androstane-17-one and 5 beta-androstane-17-one and towards the 3 beta-sulfates of 5 alpha-androstane-17-one, delta 5-androstene-17-one, 5 alpha-pregnane-20-one, and delta 5-pregnene-20-one, all of which were 100% desulfated. No sulfatase activity was found towards the 17-sulfate esters of beta-estradiol or delta 4-androstene-3-one-17 alpha-ol. The nonsteroid arylsulfate esters paranitrophenyl sulfate, paranitrocatechol sulfate, and phenolphthalein disulfate were desulfated 70, 40, and 40%, respectively. In addition to its sulfatase activity, this strain also developed C-17 oxidoreductase activity towards the estrogens and androsta(e)nes and C-3 oxidoreductase activity towards androsta(e)nes and pregna(e)nes.     

The 2',3'-dideoxyriboside of 2,6-diaminopurine selectively inhibits human immunodeficiency virus (HIV) replication in vitro

The 2',3'-dideoxyriboside of 2,6-diaminopurine(ddDAPR) is, like 2',3'-dideoxyadenosine (ddAdo), a potent and selective inhibitor of human immunodeficiency virus (HIV) in vitro. The ddDAPR compound inhibits HIV antigen expression and HIV-induced cytopathogenicity in MT4 cells at a 50% effective dose (ED50) of 2.5-3.6 microM, as compared to 3.1-6.4 microM for ddAdo. Both compounds are endowed with a high selectivity index: 112 for ddDAPR and 139 for ddAdo. The 2',3'-unsaturated derivatives of ddDAPR and ddAdo, i.e. ddeDAPR and ddeAdo, are considerably more cytotoxic and less effective against HIV than the parental compounds. Like ddAdo, ddDAPR is only weakly inhibitory to the proliferation and DNA and RNA synthesis of a series of human B-lymphoblast, T-lymphoblast and T-lymphocyte cell lines. In contrast to ddAdo, which is rapidly deaminated by beef intestine adenosine deaminase at an initial velocity (Vi) of 145 mumol/mg protein/min, ddDAPR and ddeDAPR are poor substrates for the enzyme (Vi: 8 and 0.7 mumol/mg protein/min, respectively), which further contributes to the potential of ddDAPR as a chemotherapeutic agent against AIDS.     

Monosodium urate and calcium pyrophosphate crystals differentially activate the excitation-response coupling sequence of human neutrophils

The activation patterns of human neutrophils elicited by unopsonized monosodium urate and calcium pyrophosphate dihydrate crystals were investigated. The parameters chosen, the mobilization of calcium and the synthesis of leukotrienes, are generally accepted to be relevant to the activation of the cells and their pathophysiological roles. Both particles were found to elicit increases in cytoplasmic free calcium and leukotriene synthesis. However, the rank order of potency of these two stimuli was found to be sharply dependent on the test chosen. Monosodium urate crystals were significantly more effective than calcium pyrophosphate dihydrate crystals in terms of calcium mobilization, while the latter are more potent at inducing leukotriene synthesis. These results demonstrate that these two phagocytic particles which are related to separate inflammatory joint diseases differentially activate the excitation-response coupling sequence of human neutrophils.     

Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study

Binding of NAD coenzymes to yeast alcohol dehydrogenase (YADH) and porcine heart lactate dehydrogenase (PHLDH) was studied by hydrogen-deuterium exchange with the infrared technique. Conformational changes in the enzymes specific to the coenzymes and their fragments were observed, and the pH dependence of the exchange reaction shows that it conforms to the EX-2 scheme. In both YADH and PHLDH the magnitude of the conformational change of measured by exchange retardation is considerably larger for NAD+ than for NADH. Studies with coenzyme fragments like ADP-ribose, ADP, and AMP also highlight the lack of rigorous correlation between structural features such as charge and size and their influence on exchange behavior. Ternary complexes such as YADH-NAD+-pyrazole, PHLDH-NAD+-oxalate, and PHLDH-NADH-oxamate, which mimic the transition state, have a significantly more pronounced effect on exchange rates than the corresponding binary complexes. The outstanding feature of this study is the demonstration that in the binary enzyme-coenzyme complexes the more loosely bound NAD+ is more effective in retarding exchange than the more firmly bound NADH. These differences are attributed to the unequal structural constraints exerted by the two coenzymes upon the enzymes, which translate to unequal expenditure of transconformational work in the formation of the two complexes. The opposing variation in the free energy of binding and the transconformational work expended can be viewed as an unequal partitioning of the net free energy gain resulting from the protein-ligand interaction into a binding term and that required for conformational change.