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.     

An intronic duplication in the alanine: glyoxylate aminotransferase gene facilitates identification of mutations in compound heterozygote patients with primary hyperoxaluria type 1

Summary We report here the identification of a duplication within the first intron of the gene encoding human alanine:glyoxylate aminotransferase (AGT); this duplication is closely linked to two point mutations associated with peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1 (PH1) patients. Polymerase chain reaction amplification of regions of the AGT gene including the insertion site from individuals heterozygous for this duplication, produces allele-specific fragments of different sizes. We have taken advantage of this to identify a nonsense mutation within a non-expressed allele of a compound heterozygote PH1 patient with mitochondrial AGT.     

α-Helix-to-random-coil transitions of two-chain coiled coils: Experiments on the thermal denaturation of ββ tropomyosin cross-linked selectively at C-190

A method is described for preparation of a species of β tropomyosin that is sulfhydryl-blocked at C36 and disulfide-cross-linked at C190. Five steps are involved: (1) Rabbit skeletal muscle tropomyosin, comprising αα and αβ species, is oxidized with ferricyanide, disulfide-cross-linking both species at C190. (2) The product is treated with iodoacetamide, blocking the only remaining free sulfhydryl, i.e., C36 of the β-chains. (3) The C36-blocked, C190-cross-linked product is reduced with dithiothreitol (DTT), unfolded in urea, and α and β chains separated by ion-exchange chromatography. (4) The C36-blocked β chains are refolded by dialysis. (5) The refolded, C36-blocked ββ species are cross-linked at C190 by ferricyanide oxidation. The resulting C36-blocked, C190-cross-linked ββ product is separated from contaminating species—mostly completely blocked β-chains and multichain cross-linked molecules—by size-exclusion chromatography in denaturing (guanidinium chloride) solvent. The five-step process and the final product were monitored by titration of free sulfhydryls and by NaDodSO₄/polyacrylamide gel electrophoresis (PAGE). Thermal unfolding curves from CD are reported for the resulting pure, C36-blocked, C190-cross-linked ββ species and for its DTT-reduction product, the noncross-linked C36-blocked species. The latter shows almost the same thermal unfolding transition as intact, noncross-linked ββ species. The former shows a pretransition similar to, but larger in extent than, the one well known to occur in the analogous case of C190-cross-linked αα tropomyosin. These unfolding transitions are compared with one another and with that previously reported for doubly cross-linked (at C36 and C190) ββ species. These comparisons are made in the light of current physical models for coiled-coil unfolding equilibria. It is concluded that although no extent model is demonstrably satisfactory, any successful model must include strain at the cross-link, loop entropy, and regional nonuniformities as essential parts of the physics.     

Systematics as science: A response to Cronquist

Our reply to the commentary on cladistics presented by Cronquist (1987) is aimed at four issues:

1. the application of scientific principles in systematics;
2. the recognition that the analysis of pattern is a vital precursor to any consideration of evolutionary process. A priori judgements of evolutionary process are unnecessary for the generation of informative systematic hypotheses which are chosen for their ability to explain the patterns of character distributions rather than for compatibility with any particular preconceived ideas about evolution;
3. that phenetic concepts such as overall similarity, grades, gaps, and degree of divergence, if included in methods of phylogenetic inference, will give erroneous results. Paraphyletic and polyphyletic groups must, consequently, be rejected from systematics since they have no rational empirical basis for recognition;
4. the fact that many of the problems of phylogenetic analysis attributed by Cronquist to cladistics are common to all systematic methods but that these can be dealt with by the application of such principles as parsimony, synapomorphy, and strict monophyly.

     

Nucleotide sequence and evolution of the orangutan ε globin gene region and surrounding Alu repeats

Summary We have mapped and sequenced the globin gene and seven surrounding Alu repeat sequences in the orangutan globin gene cluster and have compared these and other orangutan sequences to orthologously related human sequences. Noncoding flanking and intron sequences, synonymous sites of , , and globin coding regions, and Alu sequences in human and orangutan diverge by 3.2%, 2.7%, and 3.7%, respectively. These values compare to 3.6% from DNA hybridizations and 3.4% from the globin gene region. If as suggested by fossil evidence and molecular clock calculations, human and orangutan lineages diverged about 10–15 MYA, the rate of noncoding DNA evolution in the two species is 1.0–1.5×10^–9 substitutions per site per year. We found no evidence for either the addition or deletion of Alu sequences from the globin gene cluster nor is there any evidence for recent concerted evolution among the Alu sequences examined. Both phylogenetic and phenetic distance analyses suggest that Alu sequences within the and globin gene clusters arose close to the time of simian and prosimian primate divergence (about 50–60 MYA). We conclude that Alu sequences have been evolving at the rate typical of noncoding DNA for the majority of primate history.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Calcium-dependent anion channel in the water mold,Blastocladiella emersonii

Summary Injection of depolarizing current into vegetative cells of the water moldBlastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about –40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near –100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr²⁺ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr²⁺. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor.     

Influence of dilution on the physical state of model bile systems: NMR and quasi-elastic light-scattering investigations

Multinuclear (1H and 31P) nuclear magnetic resonance (NMR) spectroscopy and quasi-elastic light scattering have been used to characterize molecular aggregates formed in dilute sodium taurocholate--egg lecithin solutions. When mixed micelles (1.25 g/dL) are diluted with 150 mM aqueous sodium chloride, light-scattering measurements suggest a transformation from mixed micelles to unilamellar vesicle species. Decreased 1H NMR line widths for bile salt resonances are consistent with predominance of a monomer form. The concurrent appearance of a second phospholipid choline methyl resonance indicates two types of phospholipid environment in slow chemical exchange: this behavior is consistent with small unilamellar vesicles. The appearance of bilayer vesicles in dilute model bile solutions is confirmed by addition of a lanthanide shift reagent (Pr3+), which splits the 1H or 31P head-group peak into two components with distinct chemical shift sensitivities. These mixed micelle and vesicle aggregates are also distinguished by their susceptibility to the lipolytic enzyme phospholipase A2 from cobra venom.     

Coassimilation of dietary fat and benzo(a)pyrene in the small intestine: an absorption model using the killifish

Benzo(a)pyrene (BP) was dissolved in dietary fat and fed in a single dose to killifish (Fundulus heteroclitus). Fluorescence microscopic examinations of small intestinal content and frozen sections of whole small intestine revealed that during fat digestion BP was codispersed in liquid crystalline product phases produced during lipolysis (1979. Patton, J. S., and M. C. Carey, Science. 204: 145-148) and then coabsorbed with dietary lipid followed by its reappearance in intracellular fat droplets. During the time that the absorbed fat remained in the enterocytes, BP fluorescence was initially concentrated in the intracellular fat droplets and then spread throughout the cytosol of the enterocytes. Tissue analyses showed that BP was rapidly metabolized in the intestine and transported to the gallbladder. These studies show that separation of a dissolved hydrophobic carcinogen from dietary fat occurs primarily after the fat has been digested, dispersed, absorbed, and reassembled in the enterocyte. The inability of the enterocyte to discriminate between dietary fat and dissolved carcinogenic compounds may be a partial explanation of the observed link between high fat diets and the incidence of some cancers. In vertebrates, the intestine and not the liver, appears to be the major site of metabolism of dietary polycyclic aromatic hydrocarbons (PAHs).     

Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other gram negative bacteria

Summary The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described. This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb. Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria.     

Immunochemical Characterization of Pyruvate Dehydrogenase Complex in Rat Brain

Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme-linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the alpha peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the alpha E1, beta E1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar.(ABSTRACT TRUNCATED AT 250 WORDS)