Characterisation of a haem active-site mutant of horseradish peroxidase, Phe41----Val, with altered reactivity towards hydrogen peroxide and reducing substrates.

A horseradish peroxidase variant ([F41V] HRP-C*), in which Val replaces the conserved Phe at position 41 adjacent to the distal His, has been constructed. Its composition and spectroscopic, catalytic and substrate-binding properties were compared with those of the wild-type recombinant (HRP-C*) and plant (HRP-C) enzymes. Presteady-state kinetic measurements of the rate constant for compound I formation (k1) revealed an eightfold decrease in the reactivity of the Phe41----Val variant towards H2O2, in comparison with HRP-C or HRP-C*. Measurement of the remaining rate constants, k2 and k3, for the two single-electron reduction reactions of [F41V] HRP-C with para-aminobenzoic acid as reducing substrate, showed that they were 2.5-fold and 1.3-fold faster, respectively. In contrast, analysis of data from steady-state assays with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate) as reducing substrate, showed decreased reactivity of the mutant enzyme to this compound, indicating a change in substrate specificity. Over the substrate range studied, the data for HRP-C* and for [F41V] HRP-C conformed to a simple modification of the accepted peroxidase mechanism in which a first-order step (ku), assumed to be product dissociation, becomes rate-limiting under our standard assay conditions. Calculations of rate constants from steady-state data yielded values of k1 for both enzyme forms in adequate agreement with those from pre-steady state measurements. They showed, furthermore, that both k3 for 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate) and ku were substantially decreased, fivefold and tenfold, respectively, in the mutant. Analogous to the decrease in ku, we observed a twofold increase in the affinity of the mutant variant for the inhibitor benzhydroxamic acid. The coordination-state equilibrium of the haem iron also appeared shifted towards the hexacoordinate high-spin form. These observations indicate that in addition to affecting reactivity to H2O2, mutations in the distal region and close to the haem iron also affect reactivity towards different reducing substrates, inducing perturbations in the neighbourhood of the aromatic-substrate-binding site, known to be 0.8-1.2 nm from the haem iron.     

Teratocarcinoma stem cells and early mouse embryos contain only a single major lamin polypeptide closely resembling lamin B

The nuclear lamina in adult mammalian somatic cells is composed of three major proteins, lamins A, B, and C. The expression of these proteins during the differentiation of teratocarcinomas and mouse embryogenesis is described. Embryos up to day 8 of gestation and embryonal carcinoma (EC) cells express only a single lamin species closely resembling, if not identical to, lamin B. Lamins A and/or C were detected in fertilized eggs, but disappear during the first 2-4 cleavage divisions, only reappearing in 8 day post-implantation embryos. These two lamins are absent from EC cells, but are strongly expressed in some of their derivatives. These results show that cells of the early mouse embryo do not have a functional requirement for lamins A and C and imply that the structural organization of the nucleus may change fundamentally during embryogenesis.     

Structure and expression of the herpes simplex virus type 2 glycoprotein gB gene.

The gene for glycoprotein gB2 of herpes simplex virus type 2 strain 333 was cloned, sequenced, and expressed in mammalian cells. The gB2 protein had an overall nucleotide and amino acid sequence homology of 86% with the cognate gB1 protein. However, of the 125 amino acid substitutions or deletions, only 12.5% were conservative replacements. These differences were clustered within an NH2-terminal region, a central region, and a COOH-terminal region, resulting in domains of near identity broken by small regions of marked divergence. Regions of greatest homology included a 90-amino-acid stretch starting at residue 484 and 39 amino acids spanning residues 835 to 873, which cover a rate-of-entry locus mapped to Ala-552 and a syn locus mapped to Arg-857, respectively, in gB1 by Bzik et al. (D. J. Bzik, B. A. Fox, N. A. DeLuca, and S. Person, Virology 133:301-314, 1984). Pellett et al. (P. E. Pellett, K. G. Kousoulas, L. Pereira, and B. Roizman, J. Virol. 53:243-253, 1985) mapped the mutations in three monoclonal antibody-resistant gB1 mutants between amino acids 273 and 443. These epitopes are included in a region of 98 residues identical between gB1 and gB2. The identity of this protein was verified by placing a truncated gene lacking the 303 carboxyl-terminal amino acids of gB2 into mammalian COS and CHO cells. Expression was demonstrated by immunofluorescence and radioimmunoprecipitation. This protein will be purified from the stable CHO cell lines and compared with gB1 for immunogenicity and protective efficacy in animal challenge models.     

Purification and characterization of glutathione reductase from corn mesophyll chloroplasts

Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G-4507) has been purified 200-fold by affinity chromatography using adenosine 2',5'-disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)^-1 min^-1. The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate-polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES-NaOH) buffer. The sulfhydryl reagent, n -ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s^-1. The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s^-1.     

Plant Morphological and Biochemical Responses to Field Water Deficits : II. Responses of Leaf Glycerolipid Composition in Cotton

The effects of water deficits on leaf glycerolipid composition were analyzed in two photoperiodic strains of field grown cotton (Gossypium kirsutum L.) that differ in sensitivity to drought. Leaves from plants grown under dryland conditions exhibited increased dry weight and specific leaf weight. The average midday leaf water potential in the dryland treatment decreased to −1.9 and −2.4 megapascals, respectively, for the T25 and T185 genotypes. Total leaf lipid content of plants exposed to dryland conditions was 5.9 and 7.5% of leaf dry weight for strain T25 and T185, respectively. The difference in leaf lipid content between these genotypes was caused by water deficits and was attributed to loss of both phospholipids and glycolipids in strain T25. There was no apparent loss of phospholipids due to water deficits in the T185 genotype; however, a significant loss of glycolipids was partially compensated by a 2-fold increase in triacylglycerol. No change in triacylglycerol was found between treatments in T25 leaves. Water deficit caused a significant decline in the relative degree of acylunsaturation in phospholipids and glycolipids from both genotypes; however, the double bond index for triacylglycerol increased in both genotypes. It is believed that the observed responses of leaf lipid composition to dryland conditions may be an additional criterion for characterization and selection of new drought-tolerant cotton genotypes.     

C5a induction of human interleukin 1. Synergistic effect with endotoxin or interferon-gamma

Interleukin-1 (IL-1) is a potent cytokine which possesses the ability to mediate systemic acute phase responses as well as local tissue inflammation. In these studies, we have examined the ability of C5a and C5a des Arg to induce IL-1 production in vitro. Human C5a and C5a des Arg were purified to homogeneity and were found to stimulate IL-1 release from freshly obtained human mononuclear cells into the extracellular medium. Only 2 hr of exposure to the purified complement components were necessary in order to stimulate IL-1 production. The minimal concentration of C5a required was 25 ng/ml, whereas 125 ng/ml of C5a des Arg induced comparable amounts of IL-1. This dose relationship was maintained at higher concentrations (150 ng/ml vs 750 ng/ml, respectively). That the effect was due to the anaphylatoxins themselves, and not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and employing preincubation of C5a/C5a des Arg with polymyxin B. The latter blocked a wide dose range of endotoxin-stimulated IL-1 production. However, when endotoxin was added to C5a or C5a des Arg, significant synergism in the stimulation of IL-1 production was observed, occurring at various concentrations of either agent. A similar synergism with C5a/C5a des Arg was seen with interferon-gamma. In these studies, IL-1 production was measured by bioassay employing cloned D . 10 . G4 . 1 murine T cells and by radioimmunoassay for human IL-1 beta; using C5a/C5a des Arg as stimulants, there was a high degree of correlation (r = 0.82) between the two assays. Since traumatic, infectious, and inflammatory diseases may result in the simultaneous appearance of these stimuli, the synergism described herein is likely to be clinically relevant.     

Carrier RNA enhancement of recovery of DNA from dilute solutions

A study of the use of carrier RNA to improve precipitation of DNA from dilute solutions was conducted to define the conditions which optimize DNA recovery. Replicate samples containing labeled pBR322 and increasing concentrations of commercially-available Torula yeast RNA were ethanol precipitated at -20 degrees C for 1 h in microfuge tubes obtained from various manufacturers. Nucleic acids were pelleted by centrifugation for either 5 or 30 min, dried and resuspended. Although recovery was not identical in each type of microfuge tube, in all cases the percent recovery increased when carrier was added. In most cases, extending centrifugation to 30 min did not significantly increase recovery. Recovery of unlabeled DNA's of heterogeneous molecular weight and conformation was also enhanced by the addition of carrier RNA. DNA's recovered by this method can be successfully digested with BamHI and ligated with T4 DNA ligase.