Purification and properties of the coenzyme A-linked acetaldehyde dehydrogenase of Acetobacterium woodii

Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl₂ was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a ₄₄ structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH acetaldehyde dehydrogenase - ADH alcohol dehydrogenase - CHES 2-(N-cyclohexylamino)-ethanesulfonate - DTE dithioerythritol - KP-buffer 25 mM K-PO₄, pH 7.5, containing, 4 mM DTE - MES 2-(N-morpholino)-ethanesulfonate - TAPS N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate     

A portable EAG system for the measurement of pheromone concentrations in the field

A robust and portable apparatus for the measurement of pheromoneconcentrations under field conditions has been developed. Ituses the insect antenna (Lobesia botrana Hb.) itself as a sensitiveand specific pheromone detector. Shock-proof contact with theelectrodes is maintained by fixing the antenna in a specially-shapedplexiglass holder mounted within a glass tube. This allows measurementsto be made while moving the apparatus. Continuous airflow throughthe tube is generated by a suction pump and the incoming aircan be purified by passage through a charcoal filter. This allowsto readjust the offset and to calibrate the instrument by theapplication of pheromone pulses of known concentrations. Removalof the filter allows the direct access of ambient air over theantenna which responds by generating an electro-antennogram(EAG) as a measure of the pheromone concentration. Using the calibration curve, relative pheromone concentrationsin ambient air in a vineyard can be determined. Sample measurementsfrom areas treated with artificial pheromone for pest controlare presented.     

Differentiation of somatic embryos from protoplasts isolated from embryogenic suspension cultures of Abies alba L.

Embryogenic suspension cultures of Abies alba were established using an embryogenic suspensor mass culture originating from the zygotic embryo in immature seed explants (Schuller et al. 1989). Protoplasts were isolated from the suspension material. The protoplasts were immobilized in alginate layers in order to follow the development of single protoplasts. During the first days of protoplast culture a modified Kao and Michayluk (1975) medium proved to be necessary for subsequent divisions. The formation of proembryos succeeded within 2–3 weeks when subcultured with a modified Schenk and Hildebrandt (1972) liquid medium. Light, enhanced sugar concentration, and the addition of abscisic acid led to the formation of slightly green torpedo-shaped somatic embryos after 6–8 weeks from protoplast isolation.Abbreviations ABA abscisic acid - BAP N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ESM Embryonal suspensor mass (Gupta and Durzan 1986) - KM Kao and Michayluk (1975) - LP (von Arnold and Eriksson 1977) - MES 2-(N-morpholino)ethane-sulfonic acid - NAA 1-naphthalene-acetic acid (sodium salt) - PVP polyvinylpyrrolidone - SH Schenk and Hildebrandt (1972) - Tween 80 polyoxyethylene-sorbitan-monooleate     

Identification of eight determinants in the hemagglutinin molecule of influenza virus A/PR/8/34 (H1N1) which are recognized by class II-restricted T cells from BALB/c mice.

Eight nonoverlapping regions of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8), which serve as recognition sites for class II-restricted T cells (TH) from BALB/c mice, have been identified in the form of 10- to 15-amino-acid-long synthetic peptides. These TH determinants are located between residues 110 to 313 of the HA1 polypeptide. From a total of 36 HA-specific TH clones and limiting-dilution cultures of independent clonal origins, 33 (90%) responded to stimulation with one of these peptides. The residual three TH clones appeared to recognize a single additional determinant on the HA1 polypeptide which could not be isolated, however, in the form of a stimulatory peptide. None of the motifs that have been proposed to typify TH determinants were displayed by more than half of these recognition sites. Most unexpected was the finding that none of the TH determinants was located in the ectodomain of the HA2 polypeptide that makes up roughly one-third of the HA molecule. Possible reasons for the preferential recognition of HA1 as opposed to HA2 by TH are discussed.     

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated

Summary The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 by encoding a protein of 2076 amino acids and 229980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS -subunit. The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3 end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 by and encodes a protein of 2051 amino acids and 228667 Da molecular weight.     

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

Summary The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a -Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho ⁰ strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.     

In vitro toxicity testing for antibacterials against human keratinocytes

The use of cultured human keratinocytes in an in vitro comparison of topical antibacterial toxicity for epithelial cells was examined. The complement of three assessments allows testing of epithelial migration, growth, and survival. The three assessments included (1) flow cytometry for determination of cell survival, (2) a comparison of confluent cell culture growth after antibacterial exposures, and (3) an evaluation of cell migration using a technique of dermal explants to study radial migration. A comparative ranking of the toxicities of the various topical antibacterials was determined with the three assessments. This has confirmed anecdotal reports that many of the topical antibacterials are cell-toxic and may inhibit wound healing. This information can be directly extrapolated to the clinical setting, unlike many of the animal data for wound healing that currently exist.     

Processing of multi-dimensional NMR data with the new software PROSA

Summary The new program PROSA is an efficient implementation of the common data-processing steps for multi-dimensional NMR spectra. PROSA performs linear prediction, digital filtering, Fourier transformation, automatic phase correction, and baseline correction. High efficiency is achieved by avoiding disk storage of intermediate data and by the absence of any graphics display, which enables calculation in the batch mode and facilitates porting PROSA on a variety of different computer systems; including supercomputers. Furthermore, all time-consuming routines are completely vectorized. The elimination of a graphics display was made possible by the use of a new, reliable automatic phase-correction routine. CPU times for complete processing of a typical heteronuclear three-dimensional NMR data set of a protein vary between less than 1 min on a NEC SX3 supercomputer and 40 min on a Sun-4 computer system.     

A triple-resonance pulse scheme for selectively correlating amide1HN and15N nuclei with the1Hα proton of the preceding residue

Summary A 3D¹H–¹⁵N–¹³C triple resonance experiment is presented that contains exclusively cross peaks between the¹H^N and¹⁵N nuclei of one residue with the H of the preceding residue. The pulse sequence, designed to minimize the time coherence, is transverse on nuclei with short T₂ values. The experiment consists of coherence transfers via one-bond couplings from the H^N via N, CO, C to the H and back to the H^N for detection; it is called HN(COCA)HA. The experiment was tested on uniformly¹⁵N- and¹³C-enriched T4 lysozyme.