Age-related changes in the translocation of phosphatidate phosphohydrolase from the cytosol to microsomal membranes in rat liver

The effects of oleate, spermine and chlorpromazine were assayed in the presence or absence of 0.15 M KCl on the translocation of phosphatidate phosphohydrolase activity from cytosol to endoplasmic reticulum membranes in liver homogenates obtained from rats aged 1, 30, 60, 180 and 360 days. Marked age-associated decreases in phosphatidate phosphohydrolase distribution onto the membranes were demonstrated under nearly all conditions. In liver homogenates taken from 1-day-old rats and incubated with 0.15 M KCl, most of the enzyme was active (associated with the membranes). Physiological salt concentration (0.15 M KCl) produced a 2-fold increase of oleate-induced translocation of phosphatidate phosphohydrolase activity in liver homogenates from 1-day-old rats; it had no effect on those from 60-day-old rats, and produced a notable decline in liver homogenates obtained from 180- and 360-day-old rats. The promoting effect of spermine on oleate-induced translocation of this enzyme activity was higher in younger rats when incubated in the absence of 0.15 M KCl. Chlorpromazine did not show its usual antagonizing effect on oleate-induced translocation of phosphatidate phosphohydrolase when added to homogenates taken from 1-day-old rats. The antagonizing effect was slightly apparent in liver homogenates from 30-day-old rats and was more pronounced in those from 60-day-old rats in which the values diminished to one-half and to one-third either in the presence or absence of 0.15 M KCl.     

On the transposition of copia-like nomadic elements in cultured Drosophila cells

We studied the stability of the genomic distribution of six retrotransposon families in long-term and short-term cultures of Drosophila cells. In a subclone derived from Kc cells, no significant rearrangements were detected over an 8 year period. On the contrary, extensive reshuffling and amplification of transposon families were observed in recently established cell lines. These results show that in cultured Drosophila cells transposition appears to be restricted to the transition from the embryo to continuous cell lines.     

Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.     

Sulfate-chloride exchange transport in a glioma cell line

Transport of SO4(2-) was studied in the glioma cell line LRM55 to determine whether it is mediated by the Cl-/HCO3- exchanger or the K+/Cl- cotransporter previously described in these cells (Wolpaw, E.W. and Martin, D.L. (1984) Brain Res. 297, 317-327). 35SO4(2-) influx was saturable with SO4(2-). External SO4(2-) stimulated 35SO4(2-) efflux, indicating an exchange mechanism. External Cl- was a competitive inhibitor of 35SO4(2-) influx. Internal Cl- stimulated 35SO4(2-) influx and external Cl- stimulated 35SO4(2-) efflux, indicating that Cl- is an exchange substrate for the SO4(2-) carrier. Also, SO4(2-) flux was sensitive to SITS, DIDS and furosemide. However, saturating external SO4(2-) did not inhibit 36Cl- influx and did not inhibit 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 35SO4(2-) influx as it does Cl- influx. These findings indicate that SO4(2-) transport into these cells is mediated by an exchange carrier distinct from both the Cl-/HCO3- exchanger and the K+/Cl- cotransporter. While Cl- is an alternative substrate for the SO4(2-) porter, this carrier is responsible for only a minor fraction of total Cl- flux in these cells.     

Changes in host cell membrane activities in response to adhesion of Neisseria gonorrhoeae

Physiological changes in host cell model membranes (intact human erythrocytes and ghosts) as a consequence of bacterial adhesion were studied with special reference to Neisseria gonorrhoeae. Membrane activities examined were transport of K+, Cl- ions, pyruvate kinase, Na-K-dependent ATPase, and cAMP. We found that K+ and Cl- transport were affected, more so in membranes with attached pilated (P+) organisms than in those with apilated (P-) isogenic strains. In N. gonorrhoeae and in several other species of gram-negative bacteria studied, hemagglutination titres were directly correlated with effects on anion transport, suggesting that perturbations in anion transport are an immediate result of adhesion. Of three P+ gonococcus strains tested, two depressed Na-K-ATPase activity in the membrane, indicating a possible effect on the Na-K pump. Pyruvate kinase activity associated with the membrane appeared to be stimulated by attached gonococci, again by P+ strains to higher levels than P- organisms. Clearly, some enzyme properties of host membranes are intrinsically affected by bacterial adhesion. Human polymorphonuclear neutrophils were also investigated, and with some exceptions, changes observed in leukocyte enzyme activities tended to parallel those in erythrocytes. Since hypochlorous acid production is considered to be an important microbicidal mechanism in neutrophils, interference with Cl- transport could jeopardize their role in host defense.     

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba)

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].     

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.     

Symbiotic Characteristics and Rhizobium Requirements of a Leucaena leucocephala × Leucaena diversifolia Hybrid and Its Parental Genotypes

In 56-day-old plants, Leucaena leucocephala and its hybrid with L. diversifolia showed 100% more total N than did L. diversifolia. Significant (P < 0.01) host-inoculation interaction in total N was 14.4% of the total phenotypic variation. The most effective and competitive Rhizobium sp. for the leucaenas was TAL 1145. Three-strain mixed inoculation was inferior to TAL 1145 alone.     

Transcription by T7 RNA polymerase is not zinc-dependent and is abolished on amidomethylation of cysteine-347

T7 RNA polymerase has been purified to homogeneity from an overproducing clone of Escherichia coli containing pAR1219. Preparations have a zinc content as low as 0.01 mol/mol of enzyme and a high specific activity, 300 000-500 000 units/mg. There are no intrinsic zinc sites. Furthermore, extrinsic Zn2+ does not function as an activator. Supplementation of the assay mix with up to 5 mM ethylenediaminetetraacetic acid has little effect on activity while added Zn2+ is strongly inhibitory at concentrations above 10 microM. This monomeric RNA polymerase is not a zinc metalloenzyme, unlike its multimeric bacterial counterparts. Titration of the urea-denatured protein with 5,5'-dithiobis(2-nitrobenzoic acid) reveals that all 12 Cys residues are present in the free sulfhydryl form, 5 of which are readily accessible to reagent in the native enzyme. More preferential labeling of the sulfhydryls can be achieved with low concentrations of [14C]iodoacetamide, where inactivation of the enzyme proceeds with incorporation of approximately 1.2 mol of [14C]iodoacetamide/mol of polymerase. Amidomethylation primarily occurs at Cys-347, with lesser reaction at Cys-723 and Cys-839. Cys-347 and Cys-723 are in segments of the primary sequence containing numerous basic residues. These same segments have previously been implicated in promoter binding, suggesting that both residues are located within or near the active site region.     

γ-Aminobutyric Acid Receptor Complex of Insect CNS: Characterization of a Benzodiazepine Binding Site

The specific binding of [N-methyl-3H]flunitrazepam ([3H]FNZP) to a membrane fraction from the supraoesophageal ganglion of the locust (Schistocerca gregaria) has been measured. The ligand binds reversibly with a KD of 47 nM. The binding is Ca2+-dependent, a property not found for the equivalent binding site in vertebrate brain. The pharmacological characteristics of the locust binding site show similarities to both central and peripheral benzodiazepine receptors in mammals. Thus binding is enhanced by gamma-aminobutyric acid (GABA), a feature of mammalian central receptors, whereas the ligand Ro 5-4864 was more effective in displacing [3H]FNZP than was clonazepam, which is the pattern seen in mammalian peripheral receptors. The locust benzodiazepine binding site was photoaffinity-labelled by [3H]FNZP, and two major proteins of Mr 45K and 59K were specifically labelled. In parallel experiments with rat brain membranes a single major protein of Mr 49K was labelled, a finding in keeping with many reports in the literature. We suggest that the FNZP binding site described here is part of the GABA receptor complex of locust ganglia. The insect receptor appears to have the same general organization as its mammalian counterpart but differs significantly in its detailed properties.