Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor

Solvent exchange of 18O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectrometry. Buried water is labeled by equilibration of the protein in 18O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18O label eluting with the protein in 10-300 s is determined by an H2O-CO2 equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH greater than or equal to 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 18O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18O labeling of carboxyl groups at low pH. In addition to 18O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent (static accessibility greater than 0) and hydrogen-bonded main chain O, and their pH min is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate.     

Oligomerization and ring closure of immunoglobulin E class antibodies by divalent haptens

Cross-linking of antibodies constitutes a widespread initiation signal for their respective effector functions. Cross-linking IgE-class antibodies provide the triggering signal to mast cells for their degranulation process. To obtain a quantitative insight into these cross-linking processes, the interactions between a DNP-specific monoclonal antibody of the IgE class and a series of divalent DNP haptens with spacers of different length and flexibility have been studied by fluorescence titration experiments. These were analyzed by employing the theoretical model developed by Dembo and Goldstein [Dembo, M., & Goldstein, B. (1978) J. Immunol. 121, 345-353] in a fitting procedure. Equilibrium constants that describe the aggregation and ring-closure processes caused by divalent hapten binding have been used as free parameters. The intrinsic binding constants were determined by fluorescence titrations with corresponding monovalent haptens. The main results are the following: (1) The divalent haptens with a short and flexible spacer [i.e., N alpha, N epsilon-di-(DNP)-L-lysine,meso-bis[(DNP-beta-Ala)amino]succinate, and bis[(DNP-tri-D-Ala)amino]heptane, having a maximal DNP-DNP distance of gamma = 14, 21, and 45 A, respectively] effect aggregation of the antibodies mainly into closed dimers. (2) The divalent hapten family with long and rigid oligoproline spacers di(DNP)-Ahx-Asp-(Pro)n-Lys with n = 24, 27, and 33 (i.e., gamma = 100, 110, and 130 A) causes aggregation of the antibodies predominantly into closed dimers and trimers. The corresponding equilibrium constants of the respective ring-closure processes decrease significantly with longer spacer length. (3) Evidence was found that intramolecularly monomeric ring closure of the IgE antibodies is caused by haptens containing oligoproline spacers with n = 37 or 42 (gamma = 130-150 A). The equilibrium constant of the ring-closure process increases with spacer length. This increase in stability indicates a difference in the imposed strain. Furthermore, the latter results imply that the distance between the two binding sites of the IgE molecule lies in the range dictated by the rigid oligoproline part of the respective hapten's spacer, i.e., 115-130 A. (4) Nearly all oligomeric ring-closure processes proceed relatively slowly with an approximate lower limit of a half-life of 5-10 s. This slowing down of the aggregation and ring-closure processes most probably reflects steric factors.     

Lipopeptides of the N-terminus of Escherichia coli lipoprotein: synthesis, mitogenicity and properties in monolayer experiments

The N-terminal part of the lipoprotein from the outer membrane of Escherichia coli, tripalmitoyl-S-glyceryl-L-Cys-Ser and analogs with longer sequences, are polyclonal activators for B-lymphocytes. Triple-chain lipopeptides also constitute efficient low-molecular-weight carrier/adjuvant systems, which can be linked to antigens to yield immunogens for antibody production without further additives. This is the first report of monolayer experiments with chemically well defined, synthetic lipopeptide mitogens with the composition of the N-terminus of an important bacterial membrane protein. Various derivatives of the lipoprotein N-terminus were synthesized. These lipopeptides differed in the length of the peptide moiety, the number of fatty acid residues, and protective groups. In order to obtain the surface areas for the lipopeptides in isotherms and hysteresis isotherms, monolayer experiments with a computer-controlled film balance were performed. To get some information about the interaction of these compounds with typical membrane lipids mixed monolayers were formed from triple-chain lipopeptides with dipalmitoylphosphatidylcholine and cholesterol. A comparison of the mitogenic response of the compounds was made in an in vitro system with B-lymphocytes from Balb/c mice.     

Structural elements of bactopterin from Pseudomonas carboxydoflava carbon monoxide dehydrogenase

Bactopterin is a novel pterin occurring in bacterial molybdoenzymes as the organic portion of the molybdenum cofactor. Its structure is investigated here. The compound contains a single pterin ring and carries a side chain at carbon atom 6 of the pterin nucleus as indicated by the formation of pterin-6-carboxylic acid upon alkaline permanganate oxidation. Studies with phosphate-cleaving enzymes revealed the presence of two monophosphoric acid monoesters. The affinity of reduced bactopterin for thiol-Sepharose points to the presence of thiol(s) in active bactopterin.     

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200-300 micrograms/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-L-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), but not poly(dI-dC).poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the 'de novo' activity of the enzyme.     

Contribution of leaves of different ages to plant carbon balance as affected by potassium supply and water stress

The carbon balances of whole, 21-d old French bean plants (Phaseolus vulgaris L.) grown in standard nutrient solution (1K) and its modifications without (OK) or surplus (2K) potassium were calculated from the daily photosynthetic carbon inputs of individual leaves, and the daily respiratory carbon losses by individual leaves, stalks and petioles, and roots. Under the three K concentrations, maximum net photosynthetic rates (P_n) were found in the 2nd or in the 3rd trifoliate leaves, maximum respiratory rates (R_d) in the youngest, 4th trifoliate leaves; the P_n/R_d ratio decreased with leaf age. In all leaves of 2K plants, leaf dry masses and thicknesses, P_n, P_n/P_d ratios, and stomatal and intracellular conductances were lower than in OK and IK plants. Daily whole-plant net carbon gain was highest in IK plants, whereas in OK and 2K plants it was 98.0 and 81.3 % of IK, respectively. Similar values were found in the parameters of growth analysis, namely in net assimilation rates and relative growth rates. No differences were found in water potential (Ψ _w ) or water saturation deficit (W_sat) in the OK, 1K and 2K plants sufficiently supplied with water or during wilting and resaturation. The decrease in Ψ_w to −0.97 MPa was associated with a 19.9 %, 31.4 % and 23.4 % decrease in P_n of OK, 1K and 2K plants, respectively, but no effect on R_d was found. In the three variants, the short-time effect of mild water stress was fully reversible.     

HIV-specific antibody among voluntary blood donors in Lower Saxony (FRG)

Anti-HIV test results of the Red Cross Blood Transfusion Service of Lower Saxony from 1 June 1985 to 31 July 1986 inclusive were analysed retrospectively. Nine out of 70,936 donors who had not donated blood before 1 June 1985 (first-time donors) and 9 out of 261,231 donors who had donated blood before this date (repeating donors) were found anti-HIV confirmed positive at the time of the first blood donation during the study period. The prevalence of HIV antibody in first-time donors was significantly higher than in repeating donors (p less than 0.01). It was concluded that some members of risk groups used blood donation to obtain an anti-HIV test result. One out of 30,300 blood donations was confirmed anti-HIV positive. The results of this study justify the transfusion of blood donations that are reactive only in the initial ELISA test.     

Characterization of the Ss sialoglycoprotein and its antigens in Rhnull erythrocytes

The Ss sialoglycoprotein (glycophorin B) and its antigens in Rhnull erythrocytes, which lack the Rhesus blood group antigens, due to apparently silent (amorphic type) or independent suppressor (regulator type) genes, were investigated. The quantity of the molecule in amorphic and in regulator type red cell membranes was found to be decreased by about 60%-70%, as judged from sodium-dodecylsulfate polyacrylamide gel electrophoresis. The Ss glycoprotein content in the erythrocytes from heterozygotes (regulator type) was diminished to an extent of about 30%. Confirming and extending previous studies, the S, s, Ux, Uz and 'N' antigens were slightly weakened in Rhnull erythrocytes. The U and Duclos receptors were only slightly or not depressed in amorphic Rhnull cells, but almost absent from or not detectable in those of the regulator type. This demonstrates that an additional alteration, apart from the decreased Ss glycoprotein content of the membranes, accounts for the weakness of these receptors in regulator type cells. We propose the hypothesis that (a) protein(s) encoded by the Rhesus locus form(s) a complex with the Ss glycoprotein. Thus, it (they) might facilitate the incorporation of the Ss glycoprotein into the membrane and also contribute to the complete expression of the U and Duclos antigens in normal cells.     

The effects of experimental hypo- and hyperthyroidism on blood viscosity and other blood parameters in the rat

Three groups of male Sprague Dawley rats received methimazole without or with Na-thyroxine in drinking water (3 and 0.33 mg T4/l, respectively) to induce characteristic alterations of their thyroid status (hypothyroid, hyperthyroid, euthyroid). A fourth group served as an untreated control without any additive to the drinking water. With respect to the different thyroid status, the following changes in the blood parameters were found: increasing plasma-T3-levels caused a reduction in plasma viscosity, in total plasma protein and in alpha 1-globulin, but an increase in hematocrit, whole blood viscosity, the number of erythrocytes and leukocytes, alpha 2-globulin and beta-globulin. It was concluded that the increase in the plasma viscosity in the hypothyroid status is mainly due to an alteration of the plasma protein pattern, and that the increase in whole blood viscosity in the hyperthyroid rat is a consequence of increased hematocrit.