Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase.
BACKGROUND: Polyketides are important compounds with antibiotic and anticancer activities. Several modular polyketide synthases (PKSs) contain a terminal thioesterase (TE) domain probably responsible for the release and concomitant cyclization of the fully processed polyketide chain. Because the TE domain influences qualitative aspects of product formation by engineered PKSs, its mechanism and specificity are of considerable interest. RESULTS: The TE domain of the 6-deoxyerythronolide B synthase was overexpressed in Escherichia coli. When tested against a set of N-acetyl cysteamine thioesters the TE domain did not act as a cyclase, but showed significant hydrolytic specificity towards substrates that mimic important features of its natural substrate. Also the overall rate of polyketide chain release was strongly enhanced by a covalent connection between the TE domain and the terminal PKS module (by as much as 100-fold compared with separate TE and PKS 'domains'). CONCLUSIONS: The inability of the TE domain alone to catalyze cyclization suggests that macrocycle formation results from the combined action of the TE domain and a PKS module. The chain-length and stereochemical preferences of the TE domain might be relevant in the design and engineered biosynthesis of certain novel polyketides. Our results also suggest that the TE domain might loop back to catalyze the release of polyketide chains from both terminal and pre-terminal modules, which may explain the ability of certain naturally occurring PKSs, such as the picromycin synthase, to generate both 12-membered and 14-membered macrolide antibiotics. (+info
Clinical and immunochemical study of the serum IgG fraction not precipitated in a zinc-sodium salicylate reagent.
A reagent made of zinc sulphate (0-08 M) in a 0-4 M sodium salicylate solution at pH 7-3 precipitated most of the IgG when a small volume of human serum was added. Sera with normal IgG levels or polyclonal hyperglobulinaemia showed a close correlation between total IgG and zinc-precipitated IgG (r = + 0-95). In clinical material, not including IgG myeloma, zinc-soluble IgG varied between 0 and 6 mg/ml and was independent of the IgG serum concentration. In 31 normal subjects the average IgG concentration, as determined by the Technicon immunonephelometric method, was 10-2 +/- 1-7 mg/ml for total IgG and 2-2 +/- 1-0 mg/ml for the soluble fraction. Among 173 sera, including 24 from cord blood, 16 from pregnant women, and 133 from patients with miscellaneous diseases, no pathological conditions except three cases of IgG myeloma were found with a zinc-soluble IgG definitely above the normal values; zinc-soluble IgG levels were often low in patients with hyperglobulinaemia, and the difference was highly significant in liver disease. kappa and gamma light chains as well as the four IgG-Hp chain subclasses were found in both zinc-soluble fractions of normal IgG. A study of myeloma monoclonal IgG showed that globulins of classes 1, 3, and 4 could be either soluble or insoluble in the zinc reagent. One, G2, was mainly insoluble. Hexose and antistreptolysin contents per milligram normal IgG were not significantly different in either fraction. It is suggested that zinc-soluble IgG consists of the recently synthesized molecules, the zinc-solubility of which has not yet been decreased by protein association, lipid interaction, antigen binding, or enzymatic denaturation. Within this hypothesis, a low level of soluble IgG would mean either an increased precatabolic protein or a decreased synthesis. (+info
Improved antibody detection by the use of range expansion and longer filter wavelength in a low ionic strength-protamine sulphate Auto-Analyzer system.
Range expansion, achieved by insertion of a variable resistance between the colorimeter and the recorder together with the use of 550 nm colorimeter filters, has resulted in markedly improved sensitivity for antibody detection, and improved sample identification, in a low ionic strength-protamine sulphate (LISPS) system. Range expansion also permits a lower concentration of red cells to be used, thus economizing on fully typed cells. Glycerol stored frozen cells were found to be only slightly less sensitive than fresh cells in this system. (+info
Light-induced calcium influx into retinal axons is regulated by presynaptic nicotinic acetylcholine receptor activity in vivo.
Visual activity is thought to be a critical factor in controlling the development of central retinal projections. Neuronal activity increases cytosolic calcium, which was hypothesized to regulate process outgrowth in neurons. We performed an in vivo imaging study in the retinotectal system of albino Xenopus laevis tadpoles with the fluorescent calcium indicator calcium green 1 dextran (CaGD) to test the role of calcium in regulating axon arbor development. We find that visual stimulus to the retina increased CaGD fluorescence intensity in retinal ganglion cell (RGC) axon arbors within the optic tectum and that branch additions to retinotectal axon arbors correlated with a local rise in calcium in the parent branch. We find three types of responses to visual stimulus, which roughly correlate with the ON, OFF, and SUSTAINED response types of RGC reported by physiological criteria. Imaging in bandscan mode indicated that patterns of calcium transients were nonuniform throughout the axons. We tested whether the increase in calcium in the retinotectal axons required synaptic activity in the retina; intraocular application of tetrodotoxin (10 microM) or nifedipine (1 and 10 microM) blocked the stimulus-induced increase in RGC axonal fluorescence. A second series of pharmacological investigations was designed to determine the mechanism of the calcium elevation in the axon terminals within the optic tectum. Injection of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) (20 mM) into the tectal ventricle reduced axonal calcium levels, supporting the idea that visual stimulation increases axonal calcium. Injection of BAPTA (20 mM) into the tectal ventricle to chelate extracellular calcium also attenuated the calcium response to visual stimulation, indicating that calcium enters the axon from the extracellular medium. Caffeine (10 mM) caused a large increase in axonal calcium, indicating that intracellular stores contribute to the calcium signal. Presynaptic nicotinic acetylcholine receptors (nAChRs) may play a role in axon arbor development and the formation of the topographic retinotectal projection. Injection of nicotine (10 microM) into the tectal ventricle significantly elevated RGC axonal calcium levels, whereas application of the nAChR antagonist alphaBTX (100 nM) reduced the stimulus-evoked rise in RGC calcium fluorescence. These data suggest that light stimulus to the retina increases calcium in the axon terminal arbors through a mechanism that includes influx through nAChRs and amplification by calcium-induced calcium release from intracellular calcium stores. Such a mechanism may contribute to developmental plasticity of the retinotectal system by influencing both axon arbor elaboration and the strength of synaptic transmission. (+info
Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus.
A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 220.127.116.11) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family. (+info
CLIP-170 highlights growing microtubule ends in vivo.
A chimera with the green fluorescent protein (GFP) has been constructed to visualize the dynamic properties of the endosome-microtubule linker protein CLIP170 (GFP-CLIP170). GFP-CLIP170 binds in stretches along a subset of microtubule ends. These fluorescent stretches appear to move with the growing tips of microtubules at 0.15-0.4 microm/s, comparable to microtubule elongation in vivo. Analysis of speckles along dynamic GFP-CLIP170 stretches suggests that CLIP170 treadmills on growing microtubule ends, rather than being continuously transported toward these ends. Drugs affecting microtubule dynamics rapidly inhibit movement of GFP-CLIP170 dashes. We propose that GFP-CLIP170 highlights growing microtubule ends by specifically recognizing the structure of a segment of newly polymerized tubulin. (+info
Sites of reaction of pilocarpine.
Analysis of the sites of reaction of a biologically important compound, pilocarpine, a molecule with imidazole and butyrolactone rings connected by a methylene bridge, has been accomplished in a quadrupole ion trap with the aim of characterizing its structure/reactivity relationships. Ion-molecule reactions of pilocarpine with chemical ionizing agents, dimethyl ether (DME), 2-methoxyethanol, and trimethyl borate (TMB), along with collision-activated dissociation elucidated the reaction sites of pilocarpine and made possible the comparison of structural features that affect sites of reaction. Based on MS/MS experiments, methylation occurs on the imidazole ring upon reactions with CH3OCH2+ or (CH3OCH2CH2OH)H+ ions but methylation occurs on the lactone ring for reactions with (CH3O)2B+ ions. Bracketing experiments with two model compounds, alpha-methyl-gamma-butyrolactone and N-methyl imidazole, show the imidazole ring to have a greater gas-phase basicity and methyl cation affinity than the lactone ring. The contrast of methylation by TMB ions on the lactone ring is explained by initial addition of the dimethoxyborinium ion, (CH3O)2B+, on the imidazole ring with subsequent collisional activation promoting an intramolecular transfer of a methyl group to the lactone ring with concurrent loss of CH3OBO. Semiempirical molecular orbital calculations are undertaken to further address the favored reaction sites. (+info
Do charge-remote fragmentations occur under matrix-assisted laser desorption ionization post-source decompositions and matrix-assisted laser desorption ionization collisionally activated decompositions?
The precursor ions of tetraphenylporphyrins that are substituted with fatty acids can be introduced into the gas phase by matrix-assisted laser desorption ionization (MALDI) and undergo post-source and collisionally activated decompositions (CAD) in a time-of-flight mass spectrometer. The goal of the research is to obtain a better understanding of post-source decompositions (PSD); specifically, we asked the question of whether ions undergoing PSD have sufficient energy to give charge-remote fragmentations along an alkyl chain. We chose the porphyrin macrocycle because we expected it to act as an inert "support," allowing the molecule to be desorbed by MALDI and to be amenable to charge-remote fragmentation. MALDI-PSD and MALDI-CAD spectra are similar to high-energy CAD spectra and considerably more informative than low-energy CAD spectra, showing that charge-remote fragmentations of the fatty acid moieties do occur upon MALDI-PSD and MALDI-CAD. (+info