Lysosomal cystine transport. Effect of intralysosomal pH and membrane potential

The regulation of lysosomal cystine transport was studied using cystine dimethyl ester-loaded lysosomes isolated from human diploid fibroblasts. Net efflux from normal fibroblast lysosomes was compared to that from lysosomes of cystinotic fibroblasts, which contain an inherited mutation decreasing lysosomal cystine transport. This exodus of cystine from normal fibroblast lysosomes was greater than from cystinotic fibroblast lysosomes. When lysosomes were incubated with both 5 mM MgCl2 and 2 mM ATP (Mg/ATP), the amount of lysosomal cystine lost from normal lysosomes doubled, but the amount of cystine lost from cystinotic lysosomes remained small. This effect of Mg/ATP on cystine loss from lysosomes isolated from normal fibroblasts was abolished when either carbonyl cyanide m-chlorophenylhydrazone or N-ethylmaleimide was present, suggesting that the effect of Mg/ATP was mediated by the action of a lysosomal proton-translocating ATPase. Addition of KCl, RbCl, or NaCl to normal lysosomes caused smaller increases in cystine exodus. A variety of experimental conditions altered lysosomal pH, membrane potential, and the cystine lost from normal fibroblast lysosomes. These same experimental conditions produced similar alterations in the lysosomal pH and membrane potential of cystinotic fibroblast lysosomes without a comparable alteration in cystine loss. These results have led us to propose a model in which the transport of cystine out of the normal lysosome is regulated by both the lysosomal membrane potential gradient and the transmembrane pH gradient.     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.     

Characterization of three group A klebicin plasmids: localization of their E colicin immunity genes

We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin A1-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin A1-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3.     

Molecular structure of the bilin binding protein (BBP) from Pieris brassicae after refinement at 2.0 A resolution

The bilin binding protein (BBP) from the insect Pieris brassicae has been analysed for amino acid sequence, spectral properties and three-dimensional structure. The crystal structure that had been determined by isomorphous replacement has been refined at 2.0 A (1 A = 0.1 nm) resolution to an R-value of 0.20. The asymmetric unit contains four independent subunits of BBP. The co-ordinate differences are 0.25 A, in accord with the estimated error in co-ordinates. The polypeptide chain fold is characterized by an eight-stranded barrel. The connecting loops splay out at the upper end of the barrel and open it, whilst the lower end is closed. The overall shape resembles a calyx. The biliverdin IX gamma chromophore is located in a central cleft at the upper end of the barrel. The bilatriene moiety is in cyclic helical geometry with configuration Z,Z,Z and conformation syn,syn,syn. The geometry is in accord with the spectral properties and permits a correlation between sign of the circular dichroism bands and sense of the bilatriene helices. The fold of BBP is related to retinol binding protein (RBP), as had been recognized in the preliminary analysis, although the amino acid sequences of RBP and BBP show only 10% homology. There are large differences in the loops at the upper end of the barrel, whilst the segments of the centre and the lower end of the barrel superimpose closely. The ligands of BBP and RBP, biliverdin and retinol, respectively, are also similarly located.     

Pertussis Toxin Attenuates D2 Inhibition and Enhances D1 Stimulation of Adenylate Cyclase by Dopamine in Rat Striatum

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in synaptic plasma membranes isolated from rat striatum injected with pertussis toxin, which inactivates the inhibitory guanine nucleotide-binding regulatory protein (Ni) of adenylate cyclase. Pertussis toxin treatment reverted the inhibitory effects on the enzyme activity elicited by micromolar concentrations of GTP and reduced by 50% the DA inhibition of cyclase activity via D2 receptors. The toxin treatment enhanced the net stimulation of enzyme activity by DA in the presence of micromolar concentrations of GTP. However, the stimulatory effect of the selective D1 receptor agonist SKF 38393 was not significantly affected. The data indicate that Ni mediates D2 inhibition of striatal adenylate cyclase and participates in the modulation of D1 stimulation of the enzyme activity by DA.     

GC-MS identification of GA20-13-O-glucoside formed from GA20 in normal plants and dwarf-1 mutants ofZea mays L.

After feeding GA₂₀ to excised seedlings ofZea mays L. normals (N) and dwarf-1 mutants (d1), GA₂₀-13-O-glucoside (9) was identified by HPLC and by GC-MS of its permethylated derivative. The glucosylation rate of GA₂₀ was found to be higher in the dwarf-1 mutant (26%) than in the normal plant (3.6%). This article includes a GC-MS study in which diagnostic fragments from the spectra of permethylated synthetic GA glucosides have been selected that proved to be useful for the identification of permethylated GA glucosides.     

Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin

Yeast submitochondrial particles, in a P_i- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5–10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.     

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

An unstable Ring-X chromosome, Ddc⁺- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)w^vC and a Rod-X chromosome which contained a P-element mediated Ddc ⁺ insert. The resulting Ddc⁺-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc⁺-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc ⁺ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.     

Sexual dimorphism in neuronal projections from the antennae of silk moths (Bombyx mori,Antheraea polyphemus) and the gypsy moth (Lymantria dispar)

Summary The antennal lobe of both sexes of the silk moth Bombyx mori contains 55–60 ventrally located antennal glomeruli; in addition, that of the male contains a dorsal macroglomerular complex (MGC). A group of identifiable glomeruli consisting of two lateral large glomeruli (LLG) and four medial small glomeruli (MSG) is present in both sexes, but the LLG are greatly enlarged in the female. A MGC is also present in the male gypsy moth Lymantria dispar and male giant silk moth Antheraea polyphemus. The MGC in all of these species is organized into 3–4 distinct levels of glomeruli. Antennal sensory fibers were stained by cobalt backfills in B. mori, A. polyphemus, and L. dispar. Most fibers stained from cut long hairs (sensilla trichodea) projected to MGC in males and LLG in both sexes of B. mori. The distribution of fibers in the MGC of B. mori was topographically biased in that a majority of fibers from anterior branches projected medially in MGC while most fibers from posterior branches projected laterally or anteriorly. Terminal arborizations of single fibers were each restricted to a single glomerular level of the MGC. Fibers projecting to the posterior antennal center were frequently stained in cut-hair and control preparations, apparently by uptake of cobalt through intact sensilla on flagellar branches.