Crystalline aggregation of a proteolytic fragment of the major head protein of bacteriophage T4.

An analysis has been made of the composition and structure of the two types of sheets assembled from material from dissociated bacteriophage T2 (Poglazov &; Mesyhanzhinov, 1967) and T4 capsids. Serological techniques have been used to show that both types of sheet are assembled from proteolytic fragment of P23¹, the major capsid constituent. The two types of sheets have been found to interconvert depending on the concentration of Mg²⁺ ions in the buffer. Computer modelling experiments show that the “hexagonal” and “rectangular” morphologies observed in the negative stain are due to in-register and staggered associations, respectively, of a single basic hexagonal lattice. Analysis by polyacrylamide gel electrophoresis of samples of sheets and dissociated capsids, together with previous results from immune electron microscopy (Kistler et al., 1978), suggest that hexamers of the proteolytic fragment are derived conservatively from capsomers of the phage head.The value of this proteolytic P23 fragment has been twofold: (1) it has proved to be a useful peptide in the ongoing primary sequence determination of P23 and (2) antibodies raised against it have been employed to follow the fate of P23 antigenic sites during various steps of phage capsid maturation (Kistler et al., 1978).     

The penetration of local anesthetics into the red blood cell membrane as studied by fluorescence quenching.

The local anesthetics procaine and tetracaine were found to quench the fluorescence of the probes N-octadecyl naphthyl-2-amine 6-sulfonic acid and 12-(9-anthroyl)stearic acid in the presence of erythrocyte membranes. This quenching was shown to be due to the aromatic amine of the procaine and tetracaine molecules. Lidocaine, an active anesthetic that does not contain an aromatic amine in the same position as does procaine and tetracaine did not quench either of the fluorophores. The preferential quenching of the fluorescent probes by procaine and tetracaine indicated a greater accessibility of tetracaine than of procaine to the hydrocarbon region of the membrane and a greater accessibility of procaine than of tetracaine at the membrane's surface. The addition of calcium was found to reverse the quenching of 12-(9-anthroyl)stearic acid by tetracaine in the presence of red cell membranes.     

Preparation, characterization, and functions of rabbit lymph node populations. III. Antigen-induced uptake of thymidine and dibutyryl cyclic AMP: inhibition of thymidine uptake by cholera toxin and dibutyryl cyclic AMP.

Keyhole limpet hemocyanin (KLH)-primed lymph node cell (LNC) populations were incubated with various amounts of KLH and the cellular incorporation of tritiated thymidine ([³H]TdR) or tritiated N⁶, O^2′ dibutyryl cyclic AMP ([³H]DbcAMP) was determined. T LNC responded more vigorously than did complement receptor lymphocytes (CRL), i.e., B cells, at all KLH concentrations, during all time intervals examined, and in the presence or absence of normal rabbit serum (NRS). The depletion of adherent cells from KLH-primed LNC resulted in no significant decrease in KLH-induced incorporation of either [³H]TdR or [³H]DbcAMP in any of the LNC populations. Thus it appeared that variation among LNC populations in the incidence of macrophages did not account for the marked variation in their responses. Cultures containing equal numbers of T and CRL were induced to incorporate more [³H]TdR or [³H]DbcAMP than either population cultured separately or the sum of their individual responses. It was concluded that KLH-induced incorporation of these substances into primed, isolated LNC, was primarily manifested in the T-cell population. The synergism seen in cultures containing mixtures of T and CRL suggested that B cells are induced to incorporate [³H]TdR or [³H]DbcAMP in the presence of antigen and T-cell product(s). KLH-induced incorporation of [³H]TdR into KLH-primed LNC was inhibited by cholera enterotoxin (CT) and DbcAMP as previously reported. However, CT or DbcAMP inhibited this incorporation into T LNC to a greater extent than into CRL or unfractionated LNC.     

On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II.

Redox titrations of the fluorescence quenching components in chloroplasts indicate the presence of two components, one with E_m7.6 = + 25 mV and the second with E_m7.6 = -270 mV. These midpoint potentials are almost the same as those of two Photosystem II components previously shown to contribute to the chloroplast electrogenic reaction measured at 518 nm (R. Malkin, 1978, FEBS Lett.87, 329–333). Comparison of light-induced fluorescence yield changes with those obtained by redox titration suggests that both fluorescence quenchers are photoreduced. A direct demonstration of the photoreduction of the low-potential fluorescence quencher was observed in experiments at defined redox potentials. Fluorescence induction curves measured at low light intensity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) also showed a contribution from both fluorescence quenchers. An additional electron acceptor, other than the two fluorescence quenchers, was also identified in the acceptor complex. These results are discussed in terms of several electron acceptors functioning in the Photosystem II reaction center complex, and the possible function of these acceptors is considered.     

Free radical pathology: inactivation of human alpha-1-proteinase inhibitor by products from the reaction of nitrogen dioxide with hydrogen peroxide and the etiology of emphysema

Exposure of human α-1-proteinase inhibitor to 75 ppm nitrogen dioxide and 1 mM hydrogen peroxide results in a 65% loss in the anti-elastase activity of the protein after one hour. Exposure to either nitrogen dioxide or hydrogen peroxide alone has no effect. The inhibition is partially prevented when mannitol or superoxide dismutase is included during the exposure, implying that superoxide and the hydroxyl radical are formed in reactions between nitrogen dioxide and hydrogen peroxide and play a role in the inhibitory process.     

Changes in conformation and nucleotide binding of Ca, Mn, or MgG-actin upon removal of the bound divalent cation. Studies of ultraviolet difference spectra and optical rotation

The ultraviolet difference spectra of EDTA-induced denaturation of dithiothreitoltreated actin prepared with either Ca²⁺, Mn²⁺, or Mg²⁺ as the strongly bound cation showed no appreciable difference, nor could any difference be found in the change of optical rotation. However, at different wavelengths the changes in the spectra have different rates and these rates do differ significantly depending on the bivalent cation bound to G-actin. The nucleotide and the cation appear to be removed simultaneously and at the fastest rate; about 50–80% is released within 1 min. The spectral changes have two phases: a fast change whose detailed kinetics have not been investigated in this paper, followed by a slower rate with first-order kinetics. The changes of optical rotation follow a single-phase first-order kinetics. The rates depend on the divalent cation, the sequence being Mn²⁺ > Ca²⁺ > Mg²⁺. ATP release is partially reversible upon Ca²⁺ addition; the reversibility is diminished as the time of incubation with EDTA is increased. On rebinding of ATP and Ca²⁺, the spectral and optical rotatory changes are not reversed, but no further changes occur. Such an EDTA-treated actin is polymerizable after addition of Ca²⁺, and the G-actin obtained after polymerization and depolymerization shows the same spectral change on a second addition of EDTA as the original actin. On the basis of these observations a scheme is suggested for the denaturation of G-actin.     

Differences in the proteins synthesized by the progeny of the first two blastomeres of Iiyanassa, a "mosaic" embryo

Ilyanassa embryos were chemically dissociated into AB and CD blastomeres. Double-label procedures permitted detection of differences between the progeny of these two cells, in terms of the profiles of proteins synthesized approximately 4 hr after separation. The relevance of this finding to the phenomenon of cytoplasmic localization is discussed.     

Estradiol binding by intact cells isolated from DMBA-induced mammary tumors of the rat

Study of hormone binding in intact cells enables one to examine binding under conditions which elicit a biological response. Cells from 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors of the rat were enzymatically dispersed. More than 80% of these cells excluded trypan blue and were used to study binding of [3H] estradiol-17 beta. Specific binding was determined by subtracting the amount of [3H]estradiol bound in the absence and presence of 200-fold excess unlabeled estradiol. Specific binding at 37 degrees was maximal after 15 min. Steroid competition studies indicated that [3H]estradiol binding sites were relatively specific for estrogens, although there was a 9-18% inhibition of binding by androgens and progestins when present at 150-fold molar excess. Scatchard analyses of [3H]estradiol (0.15-5.0 nM) binding by whole cells suggest a single, high-affinity binding site (Kd = 7.5 x 10-10M) of low capacity (6.1 fmol/10(6) cells). More [3H]estradiol was translocated to the nucleus after 1 hr at 37 degrees than at 0 degrees. Preliminary studies indicated that incubations at 37 degrees result in appreciable metabolism of [3H]estradiol to other steroids and/or conjugates when examined by silica gel thin layer chromatography.     

The importance of different murine cell types in the immunopotentiation produced by amphotericin methyl ester.

Amphotericin R (AmB) or its methyl ester (AME) are potent stimulants of humoral and cell-mediated immunity in mice. When AME is injected simultaneously with antigen, it augments humoral immunity by expansion of the primary as well as the memory B cell pool. The most consistent and dramatic effect of amphotericin on humoral immunity is the enhancement of secondary IgG responses. This effect on the IgM-IgG switch mechanism depends on T cells and is reduced following thymectomy on adult mice. The route and timing of amphotericin administration also have dramatic quantitative, effects on immunopotentiation. Neither polyclonal B cell activation nor stimulation of antigen uptake in vitro by peritoneal macrophages was observed following intraperitoneal injection of AME. A tentative hypothesis is that antigen-stimulated T cells are important sites of the immunostimulant effects of AME on murine immune responses.     

The role of superoxide and hydroxyl radicals in the degradation of hyaluronic acid induced by metal ions and by ascorbic acid

Purified commercial hyaluronic acid contains significant amounts of iron. Addition of Fe2+ to solutions of it causes depolymerization, which is inhibited by catalase and scavengers of the hydroxyl radical (. OH) but not by superoxide dismutase. Fe3+ is ineffective. Ascorbic acid also depolymerizes hyaluronic acid, apparently because it can reduce Fe3+ in the reaction mixtures to Fe2+. Ascorbate-induced depolymerization is inhibited by the specific iron chelator desferrioxamine, by catalase, and by scavengers of the hydroxyl radical. The relevance of these observations to rheumatoid arthritis and inflammatory joint diseases is discussed.     

The inhibition and reactivation of human maternal and fetal plasma cholinesterase following exposure to the organophosphate, dichlorvos.

Spontaneous and chemically-induced reactivation of organophosphate-inhibited cholinesterase were studied using as an enzyme source plasma obtained from non-pregnant females, pregnant females at term and their respective neonates, sampled immediately following delivery. Aliquots of plasma were incubated with dichlorvos (10^?6M) for 5 min at 37°C resulting in a 96 percent inhibition of cholinesterase activity in all three groups at which time either pralidoxime chloride (10^?3M) or an equivalent volume of saline was added to the reaction flask and the restoration of cholinesterase activity was monitored over the next 120 min. Pralidoxime-mediated cholinesterase reactivation in ‘non-pregnancy’ plasma was significantly greater than that observed in either ‘maternal’ or ‘fetal’ plasma, however, no significant difference was noted in reactivation rates for these latter two groups. Significant differences were also observed in the rates of spontaneous reactivation, however, after correcting for this, there were still significant differences in the rates of pralidoxime-mediated reactivation (non-pregnant > pregnant ≥ fetal).     

Role of magnesium cations in the yeast orotate phosphoribosyltransferase catalyzed reaction. Mechanism of the inhibition by Cu++ and Ni++ ions

The magnesium chelate of the N(3)H tautomer of orotate, L₃Mg, is the true substrate in the biosynthesis of orotidine 5′-monophosphate (OMP) catalyzed by yeast orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.210) with a Michaelis constant K_m^L₃Mg equal to 12(2) μM. It is postulated that Mg⁺⁺ cations activate the transport of orotate to the active site by neutralizing the orotate charges; the ligand N(3)H is then exchanged between the incoming cation and the cation bound to the enzyme, thus ensuring the stabilization of the appropriate isomeric structure of orotate. This scheme, together with kinetic and thermodynamic data on orotate complexation by Mg⁺⁺ and Ca⁺⁺, accounts for the role of Ca⁺⁺ cations that neither activate nor inhibit OMP synthesis.Cu⁺⁺ and Ni⁺⁺ inhibiting properties arise from the formation of inert complexes of orotate. Ni⁺⁺ complexes have a poor affinity for the protein, whereas Cu⁺⁺ complexes have a Michaelis constant similar to that of the L₃Mg active species. The inertness of these complexes is tentatively understood in terms of low phosphoribosyl transfer rates as postulated from the kinetic study of the protonation of the complexes in water.     

Evidence for a synergistic salt-protein interaction -- complex patterns of activation vs. inhibition of nitrogenase by salt

The molybdenum nitrogenase enzyme system, comprised of the MoFe protein and the Fe protein, catalyzes the reduction of atmospheric N(2) to NH(3). Interactions between these two proteins and between Fe protein and nucleotides (MgADP and MgATP) are crucial to catalysis. It is well established that salts are inhibitors of nitrogenase catalysis that target these interactions. However, the implications of salt effects are often overlooked. We have reexamined salt effects in light of a comprehensive framework for nitrogenase interactions to offer an in-depth analysis of the sources of salt inhibition and underlying apparent cooperativity. More importantly, we have identified patterns of salt activation of nitrogenase that correspond to at least two mechanisms. One of these mechanisms is that charge screening of MoFe protein-Fe protein interactions in the nitrogenase complex accelerates the rate of nitrogenase complex dissociation, which is the rate-limiting step of catalysis. This kind of salt activation operates under conditions of high catalytic activity and low salt concentrations that may resemble those found in vivo. While simple kinetic arguments are strong evidence for this kind of salt activation, further confirmation was sought by demonstrating that tight complexes that have previously displayed little or no activity due to the inability of Fe protein to dissociate from the complex are activated by the presence of salt. This occurs for the combination Azotobacter vinelandii MoFe protein with: (a) the L127Delta Fe protein; and (b) Clostridium pasteurianum Fe protein. The curvature of activation vs. salt implies a synergistic salt-protein interaction.