Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 X 10(6), which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The alpha-helical and beta-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), a potent inhibitor (Ki = 1 microM) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of a phospholipase B inhibitor from Saccharomyces cerevisiae

The phospholipase B activity of plasma membrane vesicles from Saccharomyces cerevisiae is inhibited by the 100 000 X g supernatant of mechanically disrupted yeast cells. A 1850-fold purification of the inhibitor activity was achieved by gel filtration, ion exchange chromatography with DEAE-cellulose and hydrophobic interaction chromatography with Octyl-Sepharose. SDS-polyacrylamide gel electrophoresis of the purified inhibitor revealed two main bands with an apparent Mr of 60 000 and 26 500. The phospholipase B activity was strongly reduced but not completely blocked by this preparation, while the lysophospholipase and transacylase reactions, which are catalyzed by the same membrane-bound enzymes (Witt, W. et al. (1984) Biochim. Biophys. Acta 795, 108-116), were not affected.     

Translation and functional expression of cell-cell channel mRNA inXenopus oocytes

Summary mRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected intoXenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled. This coupling was due to the presence of typical cell-cell channels characterized by size-limited intercellular tracer flux, the presence of gap junctions at the oocyte-oocyte interface, and the reversible uncoupling that occurred in the presence of carbon dioxide. The induction of new cell-cell channels in the oocyte membrane was observed against a zero background or a low level of endogenous coupling, depending on the maturation stage of the oocytes. The time course of development of cell-cell coupling after the microinjection of mRNA was determined. The mRNA capable of inducing cell-cell coupling was confined to an intermediate size class when fractionated on a sucrose gradient.     

Organic free radical levels in seeds and pollen: The effects of hydration and aging

In view of their possible role in oxidative deterioration of seeds and pollen, organic free radicals were measured by electron spin resonance in embryonic axes and cotyledons of soybean [ Glycine max (L.) Merr], embryo and endosperm fractions of corn [ Zea mays L.] and pollen of cattail [ Typha latifolia L.]. A pronounced decline in the radical signal ensued when hydration increased above about 7% (wet weight basis) in both the seed materials and in pollen. Moderate hydration of the soybean axis followed by drying led to a small decrease in organic free radicals compared to untreated material, especially if the desiccation step was performed under nitrogen. In a comparison of soybeans of various ages under normal storage, organic free radical levels in the axis showed little or no increase with age. In marked contrast, over 5 days of accelerated aging at 40°C and near-saturating humidity, organic radical levels approximately doubled in the axis. This pronounced increase in free radical content was not associated with a decrease in the proportion of polyunsaturated fatty acids. The data suggest that hydration of seed and pollen causes a release of free radicals from the trapped state.     

Inhibition of geranylgeranyl diphosphate synthesis in in vitro systems

The incorporation of [¹⁴C]mevalonate and [¹⁴C]isopentenyl diphosphate into geranylgeranyl diphosphate was investigated in in vitro systems from Cucurbita pepo (pumpkin) endosperm and from Avena sativa etioplasts. Mevalonate incorporation was effectively inhibited in the pumpkin system by geranylgeranyl diphosphate and geranylgeranyl monophosphate but less effectively by phytyl diphosphate or inorganic diphosphate. Membrane lipids, geranyllinalool, or lecithin enhanced mevalonate incorporation in the Cucurbita system. Incorporation of isopentenyl diphosphate was also enhanced by lecithin and inhibited by geranylgeranyl diphosphate in the Cucurbita system. No lipid enhancement was found in the Avena system; inhibition by GGPP required a much higher GGPP concentration than in the Cucurbita system.     

Immunoreactive and biologically active somatostatin in human and sheep milk

The presence of immunoreactive and biologically active somatostatin in sheep and human milk has been demonstrated. Milk somatostatin exhibits similar chromatographic behavior to that of synthetic somatostatin-14 on both reversed-phase C18 and cation-exchange high-performance liquid chromatography columns. Milk, in contrast to plasma, contains only somatostatin-14-like material. Milk somatostatin was capable of inhibiting the basal and the prostaglandin-induced release of growth hormone from anterior pituitary cell cultures in a pattern similar to synthetic somatostatin-14. The concentrations of the peptide, as determined by radioimmunoassay, were found to be 113 pg/ml in human milk and 150 +/- 4.8 pg/ml (mean +/- range) in sheep milk. These values are severalfold higher than the corresponding concentration of the peptide in the plasma of these species. These findings are analogous to our previous observations concerning two other hypothalamic hormones, luliberin and thyroliberin [Baram, T., Koch, Y., Hazum, E. and Fridkin, M. (1977) Science (Wash. DC) 198, 300-302]. The high concentration of somatostatin and other neuropeptides in milk implies either an active concentrating mechanism in the mammary gland or an additional extrahypothalamic source for the synthesis and release of these peptides.     

Identification of the polypeptide encoded by the URF-1 gene of Neurospora crassa mtDNA

Two peptides, potentially representing antigenic determinants of a proposed gene product, were synthesized. The peptide sequences were deduced from the nucleotide sequence of the unidentified reading frame (URF)1 of the Neurospora crassa mitochondrial genome. Specific antisera to the synthetic peptides were produced. The antibodies recognized a single polypeptide species with an apparent relative molecular mass of about 30 000. The mitochondrial origin of this polypeptide was verified by in vivo labelling experiments in the presence of cycloheximide, as well as by in vitro translation using isolated mitochondria. The chemical identification of the protein was performed by partial radiosequencing of the N-terminal portion of the immunoprecipitated URF-1 product. The amount of URF-1 polypeptide present in N. crassa mitochondria is in the range of 1-2%. The protein is a constituent of the inner envelope of the organelle and probably part of a more complex membrane unit.     

d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei

Summary The new enzyme d-2-hydroxyisocaproate dehydrogenase (NAD⁺-dependent) was detected in strains of the genus Lactobacillus and related genera. Straight and branched chain aliphatic as well as aromatic 2-ketocarboxylic acids are stereospecifically reduced to the corresponding d-2-hydroxycarboxylic acids according to the following equation:R-CO-COOH + NADH + H⁺ R-CHOH-COOH + NAD⁺ The enzyme is called d-hydroxyisocaproate dehydrogenase by us because 2-ketoisocaproate is the substrate with the lowest K_M-value. NAD(H) as a cofactor cannot be replaced by NADP(H). Because of its broad substrate specificity we chose the strain Lactobacillus casei ssp. pseudoplantarum (DSM 20 008) for enzyme production and characterization. d-2-hydroxyisocaproate dehydrogenase could be purified 180-fold starting with 500 g of wet cells.The purification procedure involved liquid-liquid extraction with aqueous two-phase systems and ion-exchange chromatography. At this stage the enzyme has a specific activity of 25 U/mg and can be used for technical applications. Further purification up to a homogeneous protein with a specific activity of 110 U/mg can be achieved by chromatography on Amberlite CG 50 at pH 3.5. Properties important for technical application of the d-HicDH were investigated, especially the substrate specificity and the optimum pH- and temperature ranges for activity and stability of the catalist.     

Ultrastructural localization of the calcium-binding protein parvalbumin in neurons of the song system of the zebra finch,Poephila guttata

Summary The distribution of parvalbumin (PV) within neurons of the vocal motor nucleus hyperstriatum ventralepars caudalis (HVc) was investigated in the forebrain of adult male zebra finches by means of light and electron microscopy using the indirect immunoperoxidase technique. Parvalbumin-reaction product was located in the amorphous material of perikarya, dendrites and nuclei, and associated to microtubuli, postsynaptic densities and intracellular membranes; it was found in some axons and Gray type-2 boutons, but rarely in type-1 boutons and never in the Golgi apparatus. These observations suggest that parvalbumin may regulate calcium-dependent processes at the postsynaptic membrane and in the cytosol. Furthermore, the partial association of parvalbumin to microtubuli points to an involvement in calcium-dependent tubular functions. Calcium currents and microtubular assembly or transport may be relevant for the known functions of HVc in song learning.     

Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium

Summary The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The M_r of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing conditions three protein species (M_r: 16 500, 15 500 and 13 500) were identified in the pAF preparation. The pAF was precipitable by Ca⁺⁺ and did not cross-react with antisera against homologous purified secondary aggregation factor and lectin. It is mainly composed of protein (48.0%) and carbohydrate (50.2%). The isolated pAF restored the aggregation potency not only of factor-depleted Geodia cells but also of cells from other Demospongiae. However, the pAF displayed no aggregation enhancing effect on urea-treated cells from species belonging to the Calcispongiae or Hexactinellida. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF.