Stereospecific production of the herbicide phosphinothricin (glufosinate): purification of aspartate transaminase from Bacillus stearothermophilus, cloning of the corresponding gene, aspC, and application in a coupled transaminase process.

We have isolated and characterized an aspartate transaminase (glutamate:oxalacetate transaminase, EC 2.6.1.1) from the thermophilic microorganism Bacillus stearothermophilus. The purified enzyme has a molecular mass of 40.5 kDa by sodium dodecyl sulfate gel analysis, a temperature optimum of 95 degrees C, and a pH optimum of 8.0. The corresponding gene, aspC, was cloned and overexpressed in Escherichia coli. The recombinant glutamate:oxalacetate transaminase protein was used in immobilized form together with 4-aminobutyrate:2-ketoglutarate transaminase (EC 2.6.1.19) from E. coli for the production of L-phosphinothricin [L-homoalanin-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (AgrEvo GmbH), from its nonchiral 2-keto acid precursor 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO). In this new coupled process conversion rates of ca. 85% were obtained with substrate solutions containing 10% PPO by using only slight excesses of the amino donors glutamate and aspartate. The contamination of the reaction broth with amino acid by-products was < 3%.     

Autoradiographic comparison of [I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT_2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT_2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized ¹²⁵I-lysergic acid diethylamide ([¹²⁵I]LSD) to quantify and compare 5-HT_2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [¹²⁵I]LSD binding from 5-HT_2A and D₂ binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT_2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT_2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.     

Disruption of Potential α-Helix in the G Loop of the Guinea: Pig 5-Hydroxytryptamine2 Receptor Does Not Prevent Receptor Coupling to Phosphoinositide Hydrolysis

Abstract: Heterogeneity of the 5-hydroxytryptamine₂ (5-HT₂) receptor across species has been implicated in several pharmacological and physiological studies. Although 5-HT₂ receptors in the rat have been linked to increases in Phosphoinositide (PI) hydrolysis, little evidence exists to support the association of guinea pig 5-HT₂ receptors with Pl hydrolysis, the second messenger generally linked with 5-HT₂receptors. In the present study, we have taken a molecular and biochemical approach to determining whether species differences in brain 5-HT₂ receptors exist between rat and guinea pig. First, we isolated partial cortical 5-HT_a receptor cDNA clones that encompassed the third intracellular loop, a receptor area putatively important in receptor-effector coupling. The amino acid sequences deduced from the cDNA clones for rat and guinea pig brain 5-HT₂ receptor were 97% homologous. However, the guinea pig 5-HT₂ receptor had two tandem substitutions that disrupted a potential alpha helix in the region of the third cytoplasmic loop, which theoretically could alter the intracellular coupling of the guinea pig cortical 5-HT₂ receptor. Because of these molecular differences, we examined further the pharmacological activation of the brain 5-HT₂ receptor from guinea pig. 5-HT and the 5-HT₂ receptor agonist α-methyl-5-HT increased PI hydrolysis in guinea pig cortical slices whereas the 5-HT_1c receptor agonist 5-methyltryptamine was significantly less potent. In addition, the 5-HT₂ receptor antagonists LY53857, ketanserin, and spiperone blocked 5-HT-stimulated Pl hydrolysis. These pharmacological data suggested that activation of the 5-HT₂ receptor in guinea pig cortical slices was associated with PI hydrolysis. Thus, although areas of the guinea pig brain 5-HT₂ receptor that influence receptor-effector coupling were different from the rat, such differences were not critical to receptor-effector coupling because, as in the rat, guinea pig brain 5-HT₂ receptors were also coupled to PI hydrolysis.     

Stress and Age Related Spots with Immunoreactivity to Ubiquitin-Antibody at Protoplast Surfaces

Mesophyll protoplasts from primary leaves of 2, 3, and 4 weekold Viciafaba L. plants and from not expanded leaves of 2 weekold plants were incubated with rabbit anti-ubiquitin antibodyand FITC labeled goat anti-rabbit IgG. Dependent on age of theplant material, an increase in size and number of immunoreactivespots at protoplast surfaces were observed, when incubationswere performed after 16 h storage to allow protoplast to recover.A relationship between isolation stress and the intensity ofimmunolabeling was demonstrated for protoplasts from not expandedleaves. Furthermore, the surface of isolation stressed protoplastsshowed an increasing number of immunoreactive spots when plantswere previously exposed to water deficiency conditions for 1,2 or 4 days. Water deficiency conditions and isolation stressare therefore thought to induce ubiquitination of surface locatedproteins. A phenomenon, which seemed to be normally correlatedwith early events of senescence. (Received October 28, 1993; Accepted February 21, 1994)     

Effects of testosterone on a sexually dimorphic frog muscle: Repeated in vivo observations and androgen receptor distribution

In the present study the sexually dimorphic, androgen-sensitive flexor carpi radialis muscle (FCR) in male Xenopus laevis was viewed repeatedly in vivo to assess the influence of testosterone on muscle fiber size over a period of up to 12 weeks. Regions of the muscle innervated by different spinal nerves responded differently to testosterone treatment. Muscle fibers innervated by spinal nerve 2 (SN2) hypertrophied within 7 days in frogs that had been castrated and given testosterone-filled implants. This initial hypertrophy was followed by a return to normal fiber size a week late, after which fiber size slowly increased again. In castrated males with empty implants, muscle fibers innervated by SN2 gradually atrophied. Fibers innervated by spinal nerve 3 (SN3) were not affected by androgen replacement or withdrawal. The sartorius, a control muscle that is neither sexually dimorphic nor particularly androgen sensitive, was also unaffected. The in vivo observations were confirmed by measurements of muscle fiber cross-sectional areas in frozen sections of whole forelimbs. At 8 and 12 weeks after castration, cross-sectional areas of fibers innervated by SN2 were significantly larger in frogs provided with testosterone than in castrates without testosterone. No difference was found in the SN2 region or in the anconeus caput scapulare (triceps), another control muscle. Immunocytochemistry employing an antibody against the androgen receptor (AR) indicated that the receptor is present in myonuclei of all muscles of the forelimb. While no difference in labeling intensity was detected, the number of AR-containing nuclei per muscle fiber cross-section was higher in fibers innervated by SN2 than in those innervated by SN3, and was yet lower in the triceps. This suggests that regulation of androgen sensitivity may occur via muscle fiber. ARs, although an influence of the nerve may also contribute. 1994 John Wiley & Sons, Inc.     

Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH, NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8-9 U mg^-1 protein (µmoles mg^-1 min^-1) for NADPH and 3-4 U mg^-1 protein for NADH formation with reduced methylviologen (MV⁺⁺) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg^-1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.

The K_m-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.

Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg^-1 protein of an NADP⁺-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV⁺⁺ regeneration. The regeneration of MV⁺⁺ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg^-1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)⁺ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.

Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (D_s-(+)-isocitrate), L_s-(-)-isocitrate and 6-phosphogluconate.     

Detection and localization of triadin in rat ventricular muscle

Summary Dyads (transverse tubule—junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M_r 102K Ca²⁺ ATPase were associated with a diffuse protein band (22–30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M_r as triadin. Western blots of muscle microsomes from preparations which had been treated with 100mm iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of Mr 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.     

Formation of acetyl-CoA in Zymomonas mobilis by a pyruvate dehydrogenase complex

In the gram negative, obligately ethanologenic bacterium Zymomonas mobilis a pyruvate dehydrogenase complex was identified and the complex was enriched from cell extracts. This multienzyme complex is responsible for acetyl-CoA biosynthesis from pyruvate. No activities of related multienzyme complexes, 2-ketoglutarate dehydrogenase and branched chain keto acid dehydrogenase, could be detected.     

Involvement of Ubiquitin in Phosphoenolpyruvate Carboxylase Degradation

Western immunoblot analysis of protein extracts prepared from epidermal peels, whole leaves, and mesophyll protoplasts with ubiquitin and PEPCase antibodies indicated ubiquitinated PEPCase bands and degradation products only in crude extracts which have been obtained in the presence of the proteolysis inhibitors leupeptin and hemin. After ammonium sulfate precipitation and further purification, PEPCase forms were stable and not ubiquitinated. It is assumed, that only a certain part of PEPCase is degraded via the ubiquitin-dependent proteolysis.