Native Molecular Forms of Head Acetylcholinesterase from Adult Drosophila melanogaster: Quaternary Structure and Hydrophobic Character

The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100, 10-oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt-soluble fraction contained the same molecular forms as the low salt-soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.     

Amphiphilic and Nonamphiphilic Forms of Torpedo Cholinesterases: I. Solubility and Aggregation Properties

We report an analysis of the solubility and hydrophobic properties of the globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. We distinguish globular nonamphiphilic forms (Gna) from globular amphiphilic forms (Ga). The Ga forms bind micelles of detergent, as indicated by the following properties. They are converted by mild proteolysis into nonamphiphilic derivatives. Their Stokes radius in the presence of Triton X-100 is approximately 2 nm greater than that of their lytic derivatives. The G2a forms fall in two classes. Class I contains molecules that aggregate in the absence of detergent, when mixed with an AChE-depleted Triton X-100 extract from electric organ. AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which is also detectable in detergent-soluble (DS) extracts of electric lobes and spinal cord. Class II forms never aggregate but only present a slight shift in sedimentation coefficient, in the presence or absence of detergent. This class contains the AChE G2a forms of plasma and of the low-salt-soluble (LSS) fractions from spinal cord and electric lobes. The heart possesses a BuChE G2a form of class II in LSS extracts, as well as a similar G1a form. G4a forms of AChE, which are solubilized only in the presence of detergent and aggregate in the absence of detergent, represent a large proportion of cholinesterase in DS extracts of nerves and spinal cord, together with a smaller component of G4a BuChE. These forms may be converted to nonamphiphilic derivatives by Pronase. Nonaggregating G4a forms exist at low levels in the plasma (BuChE) and in LSS extracts of nerves (BuChE) and spinal cord (AChE).     

Strand targeting signal(s) for in vivo mutation avoidance by post-replication mismatch repair in Escherichia coli

Summary The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites. This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation. In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut ⁺) and deficient (mutHLS^-) strains. In mut ⁺ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold. Removal of the GATC site distal to the cat ^- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site. The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation. In the absence of a GATC site, mutL^- and mutS^- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH^- and mut ⁺ strains. This indicates that 50%–70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands. Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair     

Trans replication and high copy numbers of wheat dwarf virus vectors in maize cells.

The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartite geminivirus, was studied in cultured maize endosperm cells, and in the Black Mexican Sweet (BMS) maize cell line. Using in vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,000 ds DNA molecules per cell were observed in endosperm cells after three days. The replication protein was shown to act in trans, since the wild type gene of the shuttle vector enabled replication-deficient vectors carrying mutated genes to replicate. These properties suggest that WDV may have similar applications in plants as SV40 in mammalian cells.     

Alport syndrome: a genetic study of 31 families

Thirty one families with Alport syndrome including 3 families with associated syndromes were studied. The location of the COL4A5 gene, responsible for the Alport syndrome, was determined by linkage analysis with eight probes of the Xq arm and by a radiation hybrid panel. Concordant data indicated the localization of the Alport gene between DXS17 and DXS11. Four deletions and one single base mutation of the COL4A5 gene were detected. Homogeneity tests failed to show any evidence of genetic heterogeneity superimposed on clinical heterogeneity for ophthalmic signs and end-stage renal disease age.     

Microbial transformation of heterocyclic molecules in deep subsurface sediments

Recently attempts have been made to establish the presence and to determine the metabolic versatility of microorganisms in the terrestrial deep subsurface at the Savannah River Plant, Aiken, SC, USA. Sediment samples obtained at 20 different depths of up to 526 m were examined to determine carbon mineralization under aerobic, sulfate-reducing, and methanogenic conditions. The evolution of¹⁴CO₂ from radiolabelled glucose was observed under aerobic conditions in all sediments, whereas pyridine was transformed in 50% of the 20 sediments and indole was metabolized in 85% of the sediments. Glucose mineralization in certain sediments was comparable to that in the surface environment. Sulfate was reduced in only five sediments, and two were carbon limited. Methane production was detected in ten sediments amended with formate only after long-term incubations. The transformation of indole and pyridine was only rarely observed under sulfate-reducing conditions and was never detected in methanogenic incubations. This study provides information concerning the metabolic capability of both aerobic and anaerobic microorganisms in the deep subsurface and may prove useful in determining the feasibility of microbial decontamination of such environments.     

d-Hydantoinase from anaerobic microorganisms

Summary Of 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl--alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days. Offprint requests to: A. Morin     

Effects of direct transfer from freshwater to seawater on respiratory and circulatory variables and acid-base status in rainbow trout

Summary The effects of increased ambient salinity (35 mg · ml^-1) were studied at 1, 6, and 24 h after direct transfer of rainbow trout from freshwater to seawater. Two series of experiments were carried out successively. The first series was designed to simultaneously study all the respiratory (except Hb affinity for O₂), circulatory, and acid-base variables in each fish. In this series, fish were fitted with catheters chronically inserted into the cardiac bulbus, the dorsal aorta, and the opercular and buccal cavities. In the second series, designed to study haemoglobin O₂ affinity, fish were fitted with only a dorsal aorta catheter. The ventilatory flow ( ) was markedly increased just after transfer (by 55% at 1 h), then more moderately (by 20% at 6 h and 32% at 24 h). The initial hyperventilation peak was associated with frequent couphing motions. These ventilatory changes resulted essentially from increase in ventilatory amplitude. Initially, standard oxygen consumption (MM}O₂) decreased slightly, the moderately increased (by 12% at 24 h), so that the oxygen convection requirement ( ) increased substantially. In spite of an increased ventilation, the partial pressure of oxygen in arterial blood (P _aO₂) decreased slightly at 1 h, prior to returning to control levels, while partial pressure of carbon dioxide in arterial blood (P _aCO₂) was not significantly decreased. Gill oxygen transfer factor decreased substantially at 1 h (by 35%) then more moderately (by 7% at 1 h and 12% at 24 h). These results suggest a decrease in gas diffusing capacity of the gills. As P _aCO₂ remained approximatively unchanged, the gradual decrease in arterial pH (pH_a) from 7.94 to 7.67 at 24 h must therefore be regarded as a metabolic acidosis. The strong ion difference decreased markedly because the concentration of plasma chloride increased more than that of sodium. Arterial O₂ content (C _aO₂) gradually decreased (by 38% at 24 h) simultaneously with the decrease in pH_a, while the ratio P _aO₂/C _aO₂ increased. In parallel, seawater exposure induced a marked decrease in affinity of haemoglobin for O₂, so that at 24 h, P₅₀ was increased by 26% above the value obtained in freshwater-adapted trout. The increase in could be ascribed initially (at 1 h) to the decrease of P _aO₂ and later to a stimulation of respiratory neurons resulting from the lowered medullary interstitial pH. The decrease in C _aO₂ could be interpreted mainly as a consequence of a decreased affinity of haemoglobin for O₂, likely to be due to the blood acidosis and a predictable increase in chloride concentration within erythrocytes. Cardiac output ( ) slightly decreased at 1 h, then progressively increased by 30% at 24 h. Branchial vascular resistance increased at 1 h by 28%, then decreased by 18% of the control value at 24 h. Systemic vascular resistance decreased markedly by 40% at 24 h. As heart rate (HR) remained significantly unchanged, the cardiac stroke volume initially decreased then increased in relation to the changes in . The increase of , allowing compensation for the effect of decreased C _aO₂ in tissue O₂ supply, was interpreted as a passive consequence of the decrease in total vascular resistance occurring during seawater exposure.Abbreviations a.u. arbitrary units - C _aO₂ arterial oxygen content - pH₅₀ arterial pH at P₅₀ - C _vO₂ venous oxygen content - Hb haemoglobin - HR heart rate - Hct hematocrit - n_Hill Hill coefficient - O₂ standard oxygen consumption - P _aCO₂ arterial partial pressure of carbon dioxide - P _aO₂ arterial partial pressure of oxygen - P _vO₂ oxygen partial pressure in mixed venous blood - P₅₀ oxygen tension at half saturation of haemoglobin - P _VA, P _DA blood pressure in ventral and dorsal aorta - pH_a arterial pH - PIO₂, PEO₂ oxygen partial pressure of inspired and expired water - PO₂ oxygen partial pressure - cardiac output - SEM standard error of mean - S.I.D. strong ion difference - SV cardiac stroke volume - TO₂ gill oxygen transfer factor - U oxygen extraction coefficient - VA ventilatory amplitude - VF ventilatory frequency - VR_G, VR_S branchial and systemic vascular resistances - ventilatory flow - ventilatory oxygen convection requirement     

Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.