Botulinum Neurotoxin Light Chain Inhibits Norepinephrine Secretion in PC12 Cells at an Intracellular Membranous or Cytoskeletal Site

Botulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described "cell cracking" procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell "ghosts" which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca(2+)-activated [3H]norepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150-kDa NT and the 50-kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca(2+)-activated [3H]norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100-kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by "cell cracking." The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]-norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.     

Characterisation of a near infra-red absorption band of the Escherichia coli quinol oxidase, cytochrome o, which is attributable to the high-spin ferrous haem of the binuclear site.

The bacterial quinol oxidase, cytochrome o, is an enzyme which is highly analogous to the better known cytochrome c oxidase, cytochrome aa3, but with the important difference that it lacks the near infra-red absorbing pigment CuA. In this article we report an absorption band in the near IR spectrum of cytochrome o with a maximal absorption at 758 nm, and which is attributable to the ferrous high-spin haem. The 758 nm band has an extinction coefficient of 0.2-0.3 mM-1.cm-1 at 758-800 nm. This region in cytochrome aa3 is dominated by the CuA absorption. The 758 nm absorption is lost on addition of CO or cyanide to the reduced enzyme. The carbon monoxide compound of cytochrome o also has absorbance bands in the near infra-red, and these may be attributable to a low-spin ferrous haem compound.     

Non-linear inhibition curves for tight-binding inhibitors of dimeric ubiquinol-cytochrome c oxidoreductases. Evidence for rapid inhibitor mobility.

Steady-state electron flow through and electron delivery into isolated dimeric bc1 complex (ubiquinol--cytochrome c oxidoreductase) from Neurospora crassa and beef heart mitochondria were studied in the presence of increasing concentrations of antimycin A, funiculosin and/or myxothiazol. Parabolic or linear inhibition curves were obtained, depending upon the different quinols and inhibitors that were used. Linear curves occur when the inhibitor directly affects the rate-determining step. The most reasonable explanation for the parabolic curves is given by a fast intradimeric exchange of the hydrophobic inhibitors antimycin A, funiculosin (rate less than 500 s-1) and of myxothiazol (rate greater than 1 s-1). Using mitochondria from beef heart, the shape of the inhibition curve with antimycin A is parabolic if the quinol--O2 oxidoreductase turns over at about 300 s-1, but hyperbolic if the rate is 5 times less. The hyperbolic titration curve may be the result of both intradimeric and an additional interdimeric redistribution (rate approximately 100 s-1) of inhibitors between enzymes incorporated in a continuous phospholipid membrane. This explanation is supported by experiments with chromatophores obtained from Rhodobacter capsulatus. As recently described [Fernandez-Velasco, J. & Crofts, A. R. (1992) Biophys. J. 2, A153], cytochrome b becomes fully reoxidized within 1 s after a flash at substoichiometric concentrations of antimycin A. This kinetic of the slow reoxidation can be expressed in terms of the intradimeric and interdimeric redistribution with rate constants of about 10 s-1 and 2 x 10(6) M-1 s-1, respectively. It seems that rapid inhibitor redistribution may be a widespread phenomenon for hydrophobic inhibitors of enzymes incorporated in lipid membranes.     

Biochemical specificity of H-2M3a. Stereospecificity and space-filling requirements at position 1 maintain N-formyl peptide binding.

The maternally transmitted Ag is a cell surface product of three gene products: 1) H-2M3a (formerly Hmta), a class I MHC heavy chain; 2) beta 2-microglobulin; and 3) maternally transmitted factor (Mtf), the N-terminus of the mitochondrially encoded ND1 subunit of the reduced form of nicotinamide-adenine dinucleotide dehydrogenase. This class I molecule has been shown to be an N-formyl peptide receptor. Although the N-formyl moiety is necessary for binding to M3a, it is not sufficient. We proposed that the R group of the amino acid in position 1 plays a pivotal role in peptide binding to M3a. To test this hypothesis, analogues differing in size and stereospecificity of the R group were synthesized. Substitutions with other hydrophobic amino acids such as N-formyl phenylalanine and N-formyl valine had no significant effect on the ability of these Mtf alpha analogues to sensitize target cells (M3a, Mtf beta) to M3a, Mtf alpha-specific CTL. In contrast, the nonsubstituted, N-formylated, and N-acetylated glycyl analogues of Mtf beta bound equivalently to M3a in a peptide competition assay. Moreover, the alanine analogue bound in an N-formyl-dependent manner. To determine the limitations of the putative N-formyl pocket, peptide analogues were constructed incorporating D-isomer amino acids. When formylated D-alanine or D-methionine replaced the native methionine, these peptide derivatives did not show significant binding to M3a. Therefore, the presence of a space-filling R group (greater than hydrogen) is necessary for an antigenic peptide to bind M3a in an N-formyl-dependent manner. Additionally, the ability of M3a to discriminate between the optical forms of methionine and alanine demonstrates that this N-formyl pocket is stereospecific in its ability to bind peptide. Thus, we have defined three requirements for peptide binding to M3a: an N-formyl moiety at the amino terminus of the peptide, a space-filling R group at position 1 to maintain this N-formyl specificity, and the correct stereoisomer of the first amino acid.     

Localization of interferon-induced lupus inclusions demonstrated by computer image reconstruction of monensin-treated Daudi cells.

A structural analysis of cells that contained the interferon-alpha-induced lupus inclusions (LI) was performed using a high-voltage electron microscope to determine the exact cellular location of LI and their association with normal cell organelles. LI were induced in the human B lymphoblastoid cell line, Daudi, by culturing with the pure recombinant human leukocyte interferon, IFLrA. Just prior to harvesting, a portion of the cells was treated with monensin to selectively swell the Golgi apparatus, and thereby simplify their identification using the electron microscope. Organellar associations between LI and the outer nuclear envelope and Golgi apparatus were identified in stereopairs of 1-micron sections prepared from both cells that were not treated with monensin and those that were treated with monensin. Serial 0.25-micron sections of the monensin-treated cells were prepared, and seven arbitrarily chosen cells were examined. Each of these cells contained a single LI, and it formed throughout an endoplasmic-reticulum region that made contact with both the outer nuclear envelope and the Golgi vesicles. Reconstruction of a cell by computer from the digitized negatives of serial sections clearly illustrated these relationships. This study reports the first determination of the association between LI and the Golgi apparatus. It also identifies the presence of only one LI in every cell, and the routine association of the LI with both the outer nuclear envelope and the Golgi apparatus. The unique cell location of LI formation suggests their functioning in membrane biogenesis, the trafficking of proteins to the plasma membrane or to cytoplasmic vesicles, or the processing of proteins for secretion.     

Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin

The anticancer drugs adriamycin and daunomycin have each been crystallized with the DNA sequence d(CGATCG) and the three-dimensional structures of the complexes solved at 1.7- and 1.5-A resolution, respectively. These antitumor drugs have significantly different clinical properties, yet they differ chemically by only the additional hydroxyl at C14 of adriamycin. In these complexes the chromophore is intercalated at the CpG steps at either end of the DNA helix with the amino sugar extended into the minor groove. Solution of the structure of daunomycin bound to d(CGATCG) has made it possible to compare it with the previously reported structure of daunomycin bound to d(CGTACG). Although the two daunomycin complexes are similar, there is an interesting sequence dependence of the binding of the amino sugar to the A-T base pair outside the intercalation site. The complex of daunomycin with d(CGATCG) has tighter binding than the complex with d(CGTACG), leading us to infer a sequence preference in the binding of this anthracycline drug. The structures of daunomycin and adriamycin with d(CGATCG) are very similar. However, there are additional solvent interactions with the adriamycin C14 hydroxyl linking it to the DNA. Surprisingly, under the influence of the altered solvation, there is considerable difference in the conformation of spermine in these two complexes. The observed changes in the overall structures of the ternary complexes amplify the small chemical differences between these two antibiotics and provide a possible explanation for the significantly different clinical activities of these important drugs.     

Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ.

Pre-proparathyroid hormone is the major protein synthesized in wheat-germ extracts in response to addition of an 8-15S fraction of parathyroid RNA. The accuracy of the translation of the mRNA from parathyroid tissue was examined by analysis of the carboxyl-terminal tryptic peptide and the amino-terminal amino acid of the protein, by analysis of the size distribution of the mRNA, and by translation of the mRNA in a second cell-free extract. When 8-15S RNA was fractionated on a sucrose gradient containing formamide, RNA that supported the synthesis of pre-proparathyroid hormone was present in a single symmetrical peak, suggesting that it was homogeneous. Analyses by paper chromatography and electrophoresis of the proline-containing tryptic peptides of pre-proparathyroid hormone indicate that they are identical with the corresponding proline-containing peptides of parathyroid hormone. Because the COOH-terminal tryptic peptide of parathyroid hormone contains proline, the data indicate that the COOH termini of pre-proparathyroid hormone and parathyroid hormone are identical. Methionine from initiator [35S]Met-tRNAfMet was rapidly incorporated into pre-proparathyroid hormone by the wheat-germ extract, and a single-step Edman degradation selectively removed almost all of the initiator [35S]methionine present in pre-proparathyroid hormone. Translation of the 8-15S RNA in a cell-free extract from Krebs-II ascites cells resulted in a protein that comigrated with pre-proparathyroid hormone on sodium dodecyl sulfate-acrylamide gel electrophoresis. These data support the conclusion that the wheat-germ system accurately translates the mRNA for parathyroid hormone, and they strengthen the contention that pre-proparathyroid hormone is the initial biosynthetic product.     

Differentiation of normal human mammary epithelial cells in culture: an ultrastructural study.

An ultrastructural and cytochemical study of normal human mammary epithelial cells cultured from post-weaning breast fluids is described. Cells were examined at the time of plating and at intervals up to 28 days in culture. Three different stages in the morphological differentiation of these cells in vitro were observed: (1) the first stage was the formation of a monolayer of single cells, which occurred between days 1 and 10 in culture. The cells in this stage were not interconnected by junctional complexes and lacked Mg++- dependent ATPase activity in the plasma membranes, but did contain a large quantity of lipid and exhibited some secretory characteristics. (2) The second stage, occurring at 10 to 16 days in culture, was characterized by the formation of junctional complexes, the appearance of Mg++-dependent ATPase in the plasma membrane and a decrease in the number of dense bodies with peroxidase activity. (3) The third stage, occurring at 16 to 28 days in culture, was characterized by the formation of stratified layers of epithelial cells, which were interconnected by a larger number of desmosomes with numerous pleomorphic microfilaments. The Mg++-dependent ATPase activity in the plasma membrane was retained and the dense bodies with peroxidase activity were rarely observed at this stage. During the last seven days were prominent in the cells of the stratified layer. After 28 days in the culture, the cells began to round up and slough off the culture plate.     

Cytochrome components of plant microsomes.

The electron transport components of the microsomal fraction of cauliflower buds and mung bean hypocotyls were investigated using split-beam and dual wavelength spectrophotometry under a variety of reducing conditions. Cauliflower microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-559.5 [E'0 = +135 +/- 20 mV; lambdamax (reduced minus oxidised) = 559.5, 527 and 429 nm at 23 degrees C], cytochrome b5 [E'0 = -20 +/- 20 mV; lambdamax (reduced minus oxidised) = 556, 526 and 425 nm at 23 degrees C], cytochromes P-450 and P-420. On the basis of binding studies with ethyl isocyanide, degradation of cytochrome P-450 to P-420, redox potential, aniline binding, and relative rates of reduction by NADPH and NADH, it is suggested that the cytochrome P-450 system is analogous to that mammalian microsomes. Other components, reducible only by dithionite, may also be present. Mung bean microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-562 [E'0 = +120 +/- 20 mV; lambdamax (reduced minus oxidised) = 562, 528 and 430 nm at 23 degrees C], cytochrome b5, and a low potential component which was reducible only by sodium dithionite. No cytochrome P-450 or P-420 could be detected. A general method of analysis of the cytochromes was developed and applied to the microsomes from a variety of plant sources. The results indicate that large variations, both in type and amount of components, occur between the microsomes from different plant materials.     

Hydrodynamic diameters of RNA tumor viruses. Studies by laser beat frequency light scattering spectroscopy of avian myeloblastosis and Rauscher murine leukemia viruses.

The diffusion constants of avian myeloblastosis virus (AMV) and murine leukemia virus (MuLV) (Rauscher) suspensions in buffer and in 30% sucrose were determined by laser beat frequency light scattering spectroscopy at a series of temperatures ranging rom 5 to 25 degrees. By the use of the Stokes-Einstein equation, the following hydrodynamic diameters are calculated at 20 degrees: MuLV, 154 plus or minus 3 nm in sucrose and 145 plus or minus 7 nm in buffer; AMV, 144 plus or minus 3 nm in sucrose and 138 plus or minus 4 nm in buffer. While the diameters measured in buffer were temperature independent, the diameters measured in sucrose decreased by about 20% as the temperature was raised from 5 to 25 degrees. The concentration of virus particles in the suspensions ranged from 10 7 to 10 9 particles/ml. The absolute particle concentrations are estimated within plus or minus 30% by determining the dilution needed to reach a concentration sufficiently low that the particle number fluctuation contribution was comparable to that of the interference scattering. Particle weights of 3.9 x 10 8 daltons for MuLV and 4.0 x 10 8 daltons for AMV were calculated from the diffusion constants and from our own experimentally determined sedimentation coefficients. From these particle weights and the hydrodynamic diameters of the viruses, we calculated the per cent of the hydrodynamic volume of the viruses which could be freely penetrated by water, viz., 57% for AMV and 69% for MuLV.