The effect of temperature on the acetylcholinesterase activity of muscle microsomes

Membrane vesicles which constitute the sarcotubular system were separated and the fraction enriched in T-tubules purified by a calcium loading procedure. The preparations of unfractioned microsomes and T-tubules have been analyzed for their relative content of enzyme markers and acetylcholinesterase. The amount of this enzyme in the T-tubule fraction was higher than in mixed microsomes but less than two-fold the value of vesicles derived from sarcoplasmic reticulum. Arrhenius plots of membrane-bound and soluble acetylcholinesterase from either mixed microsomes or fractions enriched in T-tubules show an anomalous behaviour as two break points were obtained. The first discontinuity was found at about 17 degrees C for membrane-bound, and 12-14 degrees C for soluble acetylcholinesterase. The second one being at about 25 degrees C for both particulate and detergent-solubilized enzyme. The changes in activity with temperature suggest that lipid-protein, detergent-protein and protein-protein interactions might be involved in the stabilization of the enzyme both in the natural membrane and in the soluble state.     

Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5''-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate.     

Absence of female-specific protein in the hemolymph of stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Changes, during the reproductive cycle, in fat body, hemolymph, and ovarian proteins of the stable fly Stomoxys calcitrans were characterized quantitatively and qualitatively using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein content of all three tissues increased after blood feeding. Fat body protein increased first, followed by hemolymph and ovarian proteins. SDS-PAGE failed to identify vitellogenin in both female hemolymph and fat body samples. No single protein or group of proteins predominated at any stage of the reproductive cycle. Comparisons between male and female stable fly hemolymph and fat body proteins failed to detect female-specific proteins. Female-specific proteins, however, were detected in the hemolymph of four other species of Diptera.     

Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN-alpha (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon-treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut-off of host protein synthesis in interferon-treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut-down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut-off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon-treated cells and this early gene expression is necessary to block cellular protein synthesis.     

Some Properties of Thiosulfate-Oxidizing Enzyme from Marine Heterotroph 16B

Thiosulfate-oxidizing enzyme has been demonstrated in cell-free extracts of the marine, thiosulfate-oxidizing pseudomonad strain 16B. The enzyme, partially purified by ion-exchange chromatography and calcium phosphate gel treatment, catalyzed the oxidation of thiosulfate to tetrathionate with the concomitant reduction of ferricyanide. Native but not mammalian cytochrome c was also reduced by the enzyme in the presence of thiosulfate. The enzyme was located exclusively in the supernatant of ultracentrifuged cell extracts. The most purified enzyme preparation, like intact cells, exhibited a temperature optimum of 30 to 31°C. However, it exhibited no definite pH optimum. At pH 6.1 to 6.3 and 30°C, the K_m for thiosulfate was 1.57 mM. At lower temperatures, the apparent K_m for thiosulfate increased, but the apparent maximum velocity remained virtually unchanged. Thiosulfate oxidation in intact cells exhibited an increase in the pH optimum at lower temperatures. The thiosulfate-oxidizing enzyme of marine heterotroph 16B is compared with thiosulfate-oxidizing enzymes from other bacteria, and the effect of temperature on the relationship between pH and thiosulfate oxidation is discussed with reference to the natural habitat of the bacterium.     

Disturbance and contrasting patterns of population structure in the brachiopod Terebratulina septentrionalis (Couthouy) from two subtidal habitats

The population structures of Terebratulina septentrionalis (Couthouy) from exposed upper rock surface and semi-cryptic rock wall habitats at 33 m depth in the Gulf of Maine differ. Over a 3-yr period, population densities were consistently higher in rock wall habitats. Although both populations were dominated by juveniles (1–4 mm shell length), size-frequency distributions constructed from upper rock surface and rock wall populations were significantly different, as a result of a greater frequency of large brachiopods (> 20 mm shell length) in rock wall populations. Prominent modes occurred at 14–15 mm shell length in upper surface populations and at 19–20 mm length in rock wall populations. Recruitment was higher in rock wall habitats where ambient light intensities were significantly lower than on upper rock surfaces. Differences in recruitment are either the result of larval selection for shaded rock walls or differential juvenile mortality between habitats. The larvae of Terebratulina settle on a diverse array of substrata. These include bedrock, sandy polychaete tubes and algae in upper surface habitats and bedrock, calcareous polychaete tubes, and ascidians in rock wall habitats. Individuals attached to polychaete tubes and algae in upper surface habitats do not attain large body size (> 13 mm shell length). It is suggested that these differences in population structure reflect the greater intensity of disturbance in upper surface habitats. For example, the cod, Gadus morhua (Linnaeus), ingests brachiopods attached to algae and polychaete tubes in this habitat. Gastropod predation affects brachiopods in upper surface habitats but not in rock wall habitats. Predation by gastropods and asteroids is not size-specific. These results are consistent with the hypothesis that predation contributed to the decline in the abundance and diversity of articulate brachiopods since the Mesozoic, and suggest that the restriction of recent populations to semi-cryptic rock wall and crevice habitats is, in part, controlled by disturbance.     

Enhancement of susceptibility of HSV-1-infected cells to natural killer lysis by interferon

The effect of interferon (IFN) on the natural killer (NK) activity of human PBL against HSV-1-infected HeLa cells was studied. Human PBL from several individuals did not consistently show a preferential lysis of HSV-1-, vaccinia-, or adenovirus type 5-infected cells with respect to uninfected HeLa cells. Treatment with IFN of effector PBL increased their lytic activity but did not alter the degree of preference on the lysis of the target cells shown by untreated PBL. Pretreatment with IFN of HSV-1-infected HeLa cells increased their susceptibility to lysis 5- to 10-fold. In contrast, identical pretreatment of the uninfected, adenovirus type 5- or vaccinia virus-infected HeLa cells before the assay decreased their susceptibility to NK lysis. This effect was not likely to be due to a block of the viral replication because other inhibitors like mitomycin C did not have the same effect. All target cells induced IFN synthesis in effector PBL cells. A similar level of IFN was induced by HSV-1-infected or uninfected HeLa cells. Pretreatment with IFN of HSV-1-infected, but not uninfected, HeLa cells induced 5 to 10 times more IFN by PBL, in good correlation with the increase in lytic activity. PBL treated with IFN, however, in conditions to give maximal stimulation of NK activity, presented the same preferential lysis of HSV-1-infected HeLa cells and synthesized similar levels of IFN as untreated PBL. In addition, HSV-1-infected HeLa cells were killed through different target structures than uninfected cells. Taken together, our results indicate an effect of IFN at the level of the NK target structures in HSV-1-infected HeLa cells by increasing either their number or, more likely, their affinity for NK cells independent of the effect of IFN in the effector cells or as an antiviral agent.     

Differential recognition of the serologically defined HLA-A2 antigen by allogeneic cytotoxic T cells

A comprehensive analysis of human alloimmune cytotoxic T lymphocytes (CTLs) specific for the HLA-A2 antigen identified 11% of HLA-A2 positive cells as outliers. In total, 11 unrelated serologically indistinguishable, but distinguishable by cell-mediated lympholysis (CML) HLA-A2 positive outlier cells were identified. The outlier cells could be subdivided in two subgroups according to reactivity patterns obtained with CTLs directed against the HLA-A2 antigen of outlier cells and their inhibitory capacity in specific competitive inhibition experiments. Thus, the serologically defined HLA-A2 specificity can be divided into at least three subtypes using CTLs specific for the HLA-A2 antigen. Moreover, CTLs specific for an HLA-A2 subtype could be induced when responder cells expressed a different HLA-A2 subtype antigen. On the basis of several family studies, we conclude that the subtype HLA-A2 antigens are inherited in a codominant way.     

Analysis of human class I antigens by two-dimensional gel electrophoresis

Class I antigens were isolated by immunoprecipitation from cell extracts prepared from mitogenically stimulated and internally radiolabeled peripheral blood lymphocytes (PLBs). The precipitating antibodies used are monomorphic and recognize a determinant on the heavy chain of HLA-A, B, C antigens regardless of their allelic specificities when complexed with ₂m, or determinants on ₂m itself. Comparison of class I molecules isolated from 25 different homozygous typing cels (HTC) and analyzed by two-dimensional (2-D) gel electrophoresis allowed the identification of those HLA-A,13 locus specificities most common in the European Caucasoid population. Class I antigens isolated from HTC that are HLA identical are biochemically indistinguishable also. Evidence was obtained for the expression of additional class I antigens besides the HLA-A, B, C locus products: for some haplotypes, up to six class I genes may be active in mitogenically activated PBLs. No differences in molecular weight and isoelectric point of the class I heavy chains were observed between the antigens recognized by W6/32, the anti-heavy chain reagent, and anti- ₂m reagents. The nature of the mitogenic stimulus, i. e., pokeweed mitogen or phytohemagglutinin, was irrelevant with respect to the class I antigens isolated by this method. Using the HTCs as reference, a panel of HLA-B27 positive heterozygous cells was analyzed. Two types of HLA-B27 antigens, distinct by CML typing were represented. These two forms differed also in their biochemical properties. In addition, we obtained evidence for the existence of an A2 variant. This finding was likewise confirmed by CML typing.