Evaluation of H2O activity in the free or phosphorylated catalytic site of Ca2+-ATPase

The sarcoplasmic reticulum Ca2+-ATPase catalyses a reversible calcium transport coupled to phosphate transfer between ATP and water. It has been proposed [Biochemistry (1980) 19, 4252-4261] that the reactivity of the acyl-phosphate bond is dependent on the water activity within the catalytic site. We have tested this hypothesis and found that the polarity in the free catalytic site is lower than that of water, a further and large decrease is observed when the enzyme is phosphorylated by Pi. Phosphorylation by ATP indicates that this polarity change is specifically associated with the formation of the ADP-insensitive phosphoenzyme.     

Elevation of a threonine phospholipid in polyoma virus transformed hamster embryo fibroblasts.

Analysis of the lipids of normal hamster embryo fibroblasts and polyoma virus transformed fibroblasts shows a decrease in phosphatidylcholine and phosphatidylethanolamine and a marked increase in a threonine phospholipid after transformation. Transformed cells also react differently with fluorodinitrobenzene and trinitrobenzenesulfonate. phosphatidylethanolamine of transformed cells reacts to a greater extent with both probes. Phosphatidylserine and the threonine phospholipid of both cells do not react with trinitrobenzenesulfonate. The threonine phospholipid is provisionally identified as phosphatidylthreonine.     

Effect of N2 on the mutagenic and lethal activities of ICR-170 in Neurospora crassa

The nature of the N₂ effect for ICR-170, i.e., the greater mutagenic and lethal activities of this agent in the presence of N₂ than O₂, has been studied at the ad-3 region of Neurospora crassa. The characteristics of the N₂ effect for ICR-170 were that (1) the N₂ effect with ICR-170 was displayed in conidia when N₂ was administered during, but not before or after, ICR-170 treatment, (2) the highly increased mutagenic and lethal activities of ICR-170 in the presence of N₂ were due to an anoxic condition rather than to the presence of N₂ per se, (3) the high killing activity of ICR-170 under N₂ was due largely to increased cytoplasmic inactivation, (4) the N₂ effect was a general phenomenon at the ad-3 region of N. crassa, and (5) the highly ICR-170-induced mutation in conidia under N₂ was attributable to an actual enhancement in the mutagenic activity of ICR-170 rather than to selective killing. With regard to the mechanisms of the N₂ effect with ICR-170, results indicate that this effect (1) was not due to more extracellular oxidative degradation of ICR-170 molecules in the presence of O₂, or to a greater uptake of ICR-170 by conidia under N₂, but (2) was due to the inhibition of conidial respiration under an anoxic environment.     

Interaction of membrane aminophospholipids of E. coli with fluorodinitrobenzene and trinitrobenzenesulfonate.

E. coli cells were reacted with TNBS in bicarbonate-NaCl buffer, pH 8.5 (buffer A) and in phosphate-NaCl buffer, pH 7.0 (buffer B). In buffer A, DNP-GPE is the major product when FDNB is used. DNP-PE and DNP-LPE are formed in lesser amounts. Phospholipase A activity is high in buffer A. When TNBS is used, the labeling of the lipid components is less than with FDNB and more TNP-PE is formed relative to TNP-GPE. This data suggests that the phospholipases which are located primarily on the outer L-membrane of the cell wall act to a lesser extent on TNP-PE than on DNP-PE. E. coli cells were prelabeled with TNBS and FDNB in buffer A, washed and incubated in buffer A. The endogenous labeled DNP-PE gradually decreased with time with a concomitant increase in DNP-LPE and DNP-GPE due to phospholipase A activity. In contrast, the endogenous labeled TNP-PE also decreased with time as did the endogenous labeled TNP-LPE but a new orange lipid was produced. This lipid is believed to be a derivative of TNP-PE in which one of the nitro groups has been reduced to an amino group by nitroreductase. E. coli cells were prelabeled with TNBS and FDNB in buffer A, washed and incubated in buffer B. Under these conditions with both TNBS and FDNB there is an increase in TNP-PE and DNP-PE with a concomitant decrease in TNP-LPE, TNP-GPE, DNP-LPE and DNP-GPE. These results show that at neutral pH acylation occurs to regenerate TNP-PE and DNP-PE. E. coli cells were incubated with exogenous DNP-GPE or TNP-GPE in buffer A. The DNP-GPE and TNP-GPE were rapidly hydrolyzed by a phosphodiesterase to DNP-ethanolamine and TNP-ethanolamine. An orange derivative was formed which was provisionally identified as a derivative of DNP-ethanolamine or TNP-ethanolamine in which a nitro group has been reduced to an amino group by nitroreductase. The phospholipases and acylating enzymes present in the cell wall of E. coli are active on the dinitrophenyl and trinitrophenyl derivatives of PE and LPE and may act in concert to model and repair the plasma membrane.     

Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae

Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged. In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strain's reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2. Replacement of the conserved His(174) with alanine within the C-terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro. Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth. Deletion mutants further revealed that the N-terminal region of MetAP2 (residues 2-57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth. Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N-terminal region to interfere with wild-type MetAP2 function.     

A global but stable change in HeLa cell morphology induces reorganization of DNA structural loop domains within the cell nucleus

DNA of higher eukaryotes is organized in supercoiled loops anchored to a nuclear matrix (NM). The DNA loops are attached to the NM by means of non-coding sequences known as matrix attachment regions (MARs). Attachments to the NM can be subdivided in transient and permanent, the second type is considered to represent the attachments that subdivide the genome into structural domains. As yet very little is known about the factors involved in modulating the MAR-NM interactions. It has been suggested that the cell is a vector field in which the linked cytoskeleton-nucleoskeleton may act as transducers of mechanical information. We have induced a stable change in the typical morphology of cultured HeLa cells, by chronic exposure of the cells to the polar compound dimethylsulfoxide (DMSO). Using a PCR-based method for mapping the position of any DNA sequence relative to the NM, we have monitored the position relative to the NM of sequences corresponding to four independent genetic loci located in separate chromosomes representing different territories within the cell nucleus. Here, we show that stable modification of the NM morphology correlates with the redefinition of DNA loop structural domains as evidenced by the shift of position relative to the NM of the c-myc locus and the multigene locus PRM1 --> PRM2 --> TNP2, suggesting that both cell and nuclear shape may act as cues in the choice of the potential MARs that should be attached to the NM.     

Analysis of spontaneous gene conversion tracts within and between mammalian chromosomes

In the present study, we report the first characterization of gene conversion tract length, continuity and fidelity for pathways of gene targeting, ectopic and intrachromosomal homologous recombination using the same locus and mammalian somatic cell type. In this isogenic cell system, the vast majority of recombinants (> 97%) are generated by homologous recombination and display a high degree of fidelity in the gene conversion process. Individual gene conversion tracts are highly likely to involve single, independent recombination events and proceed through a heteroduplex DNA intermediate. In all recombination pathways, gene conversion tracts are long, extending up to ∼ 2 kb. Most gene conversion tracts are continuous in favor of donor region sequences, but in a small fraction of recombinants (15%), discontinuous gene conversion tracts are observed. In most cases, the recombination donor sequence is unaltered, although in two cases of intrachromosomal recombination, both recombination donor and recipient sequences bear gene conversion tracts. Overall, gene conversion events are similar, both qualitatively and quantitatively, for homologous recombination within and between mammalian chromosomes.     

Mechano-sensitive channels regulate the stomatal aperture in Vicia faba

In the bright fields, stomata of the plants are fully opened to raise the transpiration rate and CO₂ uptake required for photosynthesis. Stomatal opening is driven by the activation of plasma membrane H⁺-ATPase and K⁺_in channels, and the Ca²⁺-dependent inactivation and blockage of both components were supposed to be inevitable function to regulate the stomatal aperture. Although, it is still obscure how these activities are regulated at the open state. Application of an amphipathic membrane creator, trinitrophenol (TNP), instantly generates the convex curvature in the plasma membrane, which occurs in the phases of stomatal opening and closure. TNP surely activates mechanosensitive Ca²⁺-permeable channels and attenuates the promotion of stomatal opening, but does not inhibit and promote stomatal closure. These results suggest that activation of mechanosensitive Ca²⁺-permeable channels regulates the opening phase of stomata in plants.     

FcεRI-induced mast cell cytokine production critically involves an aspartic acid residue (D234) in the C-terminal intracellular domain of the FcεRIβ chain

The high affinity IgE Fc receptor (FcεRI) β chain is well implicated as a signal amplifier through the immunoreceptor tyrosine-based activation motif (ITAM) in its C-terminal intracellular region. Our previous study, however, demonstrated that mutation in all of the three tyrosine residues within the FcεRIβ ITAM did not impair FcεRI-induced cytokine production, suggesting a possible functional region other than the ITAM. To investigate the ITAM-independent mechanism by which FcεRIβ regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIβ mutants were generated. We observed that truncation of the FcεRIβ C-terminus downstream of the ITAM resulted in a considerable decrease in FcεRI-induced IL-6 production but not degranulation. Furthermore, mutagenesis of a single C-terminal aspartic acid (D234) to alanine (β-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and β-D234A suggests that the secondary structure of the FcεRIβ C-terminus was not affected by the D234A mutation. Consistently, we did not observe any effect of this mutation on FcεRI-induced tyrosine phosphorylation of FcεRIβ. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIβ ITAM.     

Controlled coupling of mildly reduced proteins to Sepharose gelatin by heterobifunctional reagent

The heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate was utilized for controlled coupling of mildly reduced BSA and lysozyme to Sepharose gelatin to prepare immunoabsorbents. Each reaction step was examined and quantitated. The free amino groups on gelatin after coupling gelatin to cyanogen bromide-activated Sepharose as well as the number of 3-(2-pyridyldithio) propionyl groups on Sepharose gelatin were quantitated. Coupling of mildly reduced BSA as well as lysozyme to PDP-Sepharose gelatin occurred through sulfhydryl-disulfide exchange and permitted the formation of an immunoabsorbent. The immunoabsorbents were capable of binding the respective antibody and the eluted antibodies were pure and free of plasma proteins.