Reactions of phosphonate analogs of pyridoxal phosphate with apo-aspartate aminotransferase.

We have investigated reactions of the 5-phosphonoethyl and 5-phosphonoethenyl analogs of pyridoxal 5'-phosphate in the coenzyme site of cytosolic aspartate aminotransferase. Acid dissociation constants and equilibrium constants for hydration and for tautomerization have been evaluated for these compounds. In confirmation of previous results, both compounds are partially active. They bind to apoenzyme well and undergo conversion in the presence of glutamate to amine forms which show induced circular dichroism comparable to that of native enzyme. A normal "external" Schiff base is evidently formed with 2-methylaspartate, but the amounts of quinonoid intermediate formed with erythro-3-hydroxyaspartate are less than those formed with pyridoxal phosphate. The pKa of the imine group of the enzyme reconstituted with the phosphonoethyl analog is more than two units lower than that in the native enzyme. Binding of the dicarboxylates glutarate, 2-oxoglutarate, and succinate shifts the pKa upward. The absorption spectra of the resulting complexes indicate the existence of at least three low pH species. A shift of 2.3 to 2.9 ppm to a lower frequency was observed for the 31P NMR signal upon binding of these dicarboxylates or of 2-methylaspartate. Enzyme containing the analogs crystallizes. Polarized absorption spectra suggest that the coenzyme has an orientation similar to that of pyridoxal phosphate in the native enzyme.

Clavulanate inactivation of Staphylococcus aureus beta-lactamase.

The interaction of clavulanic acid with beta-lactamase from Staphylococcus aureus was investigated, particularly with a view to determining whether conformational effects are involved. The inactivation at neutral pH is essentially stoichiometric, leading to an inactive species with an enamine chromophore. Two forms of the enamine were observed, the first-formed having a positive ellipticity with a maximum near 290 nm. This species slowly converted into the stable form of the inactivated enzyme that had a negative ellipticity with a minimum at 275 nm. This change in sign of the ellipticity of the enamine is consistent with the previously proposed cis-trans isomerization of the enamine [Cartwright & Coulson (1979) Nature (London) 278, 360-361). Both the far-u.v.c.d. and the intrinsic viscosity of the inactivated enzyme indicated that negligible change in conformation of the enzyme accompanied inactivation. The rates of inactivation and enamine formation were compared at low temperatures, where the initial rates were slow enough to be monitored. The rate of loss of 95% of the catalytic activity was almost 100-fold faster than the rate of formation of the first-formed enamine species. The remaining 5% activity was lost with a rate comparable with that for formation of the initial enamine. The simplest explanation of these results is that a relatively stable acyl-enzyme intermediate builds up initially and more slowly partitions between turnover (hydrolysis) and enamine formation. The initially formed enamine is in the cis conformation but slowly isomerizes to the more stable trans form.

[In vitro and in vivo production of amines by a Lactobacillus strain isolated from a cock crop]. / Production d'amines in vitro et in vivo par une souche de Lactobacille isolée d'un jabot de coq.

The chicken digestive tract is mainly colonized by bacteria belonging to the Lactobacillus genus. One of these strains (LEM-207) isolated from the crop of a cock and closely resembling L. acidophilus, was able to develop on a carbohydrate-free medium. Production of carbon dioxide and synthesis of tyramine, putrescine and cadaverine were observed in the cultures. Once implanted in the crops of germ-free chickens, strain LEM-207 led to the formation of amines. In germ-free (axenic) animals, only endogenous tyramine was detected, whereas in monoassociated chickens, we found a production of tyramine, cadaverine and putrescine. The concentrations of cadaverine and putrescine decreased with increasing acidification of the contents, whereas the level of tyramine increased (7-fold higher level than in germ-free chicken). Amine production was not detected in the caeca. The toxicological aspects of tyramine production in terms of the animal are discussed.

Role of calmodulin in neurotransmitter synthesis.

Tyrosine 3-monooxygenase (EC 1.14.16.2) and tryptophan 5-monooxygenase (EC 1.14.16.4) are generally believed to be the rate-limiting enzymes in the biosynthesis of the neurotransmitters, catecholamines and serotonin, respectively, and therefore the regulation of their activities is of particular importance. At least three calmodulin-dependent protein kinases differed in their molecular weights and substrate specificities, designated I, II, and III in the order of decreasing molecular weight, in rat brain cytosol. Among them, calmodulin-dependent protein kinase II with a molecular weight of about 540,000 appeared to occur only in the nervous tissues. Kinase II was found, on the one hand, to phosphorylate tyrosine 3-monooxygenase and tryptophan 5-monooxygenase, leading to the activation of these monooxygenases in the presence of activator protein and, on the other hand, to phosphorylate tubulin and microtubule-associated protein 2, which results in disassembly of the microtubules that had been assembled. These results suggest the possibility that both the secretion and biosynthesis of monoamine neurotransmitters stimulated by Ca2+ influx in the nervous system may be regulated by calmodulin-dependent protein kinase II via the phosphorylation of microtubule proteins and the phosphorylation of the monooxygenases that are the rate-limiting enzymes in the biosynthesis of the neurotransmitters.

Frequency-pulsed electron capture gas-liquid chromatographic analysis of metabolites produced by Clostridium difficile in broth enriched with amino acids.

Clostridium difficile strain CDC A-567 was cultured in Trypticase (BBL Microbiology Systems)-yeast-salt broth supplemented with 0.2% L-leucine, L-norleucine, L-isoleucine, L-tyrosine, or L-tryptophan. Four extractions were done on the spent medium, three at pH 2 and one at pH 10, using CHCL3 or ether. Derivatizations were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol. All samples were analyzed with frequency-pulsed electron capture gas-liquid chromatography. A dedicated computer was used to assist in data analysis. C. difficile produced both short-chain and aromatic acids in Trypticase-yeast-salt broth; hydroxy acids were also detected. p-Cresol, indoleacetic acid, 4-methylthio-2-hydroxybutyric acid, and some unidentified alcohols were observed. The basic chloroform extraction contained cadaverine and putrescine. Leucine, norleucine, and isoleucine influenced the production of C5 and C6 acids and alcohols. L-Tyrosine underwent successive degradation to produce p-cresol and aromatic acids as final products. Tryptophan increased the production of indoleacetic, indolepropionic, and indolebutyric acids. Isocaproic acid was produced in relatively high concentrations regardless of medium substitution. The consistent production of iC6 under various substrate conditions indicates that the production of this compound might be consistent enough in vitro to form the basis of a rapid test for detection of C. difficile in stool specimens by frequency-pulsed electron capture gas-liquid chromatography.

Biosynthesis of some urinary trace amines in the rat and the human.

The biosynthesis and urinary excretion of the trace amines PE, mTA, pTA and TRYP have been investigated after systemic injection in the rat and ingestion in the human. In the rat the likely precursors phe, tyr, DOPA and PE, radioactively labelled, were injected in tracer and supplemented doses. pTA was shown to be formed from PE and ptyr and in tiny amounts from phe and DOPA. mTA was formed from PE, phe and DOPA. PE was found after phe and PE injections. In the human, after ingestion of the deuterated precursors phe, ptyr, PE and try, only deuterated PE and mTA were located after phe, deuterated pTA after ptyr, deuterated PE after PE and deuterated TRYP after try. These results suggest that in the periphery pTA may be formed by p-hydroxylation of PE as well as by decarboxylation of ptyr while PE and TRYP are formed by decarboxylation of their parent amino acids phe and try respectively. PE is also a possible source of mTA but in this case hydroxylation and decarboxylation of phe and dehydroxylation and decarboxylation (or vice versa) of DOPA are also important.

Biogenic monoamines in developing taste buds of mouse circumvallate papillae.

After administration of monoamine precursors, developing taste buds of newborn and young mice were observed by means of electron microscopy and fluorescence histochemistry. Gustatory (type III) cells occurred in the primitive taste buds during stage 1 (0-1 day after birth). These cells had an immature type of afferent synaptic contacts with nerve terminals; however, no specific fluorescence was found in the taste buds after administration of 5-HTP or L-DOPA. During stage 2 (2-7 days), mature types of afferent synapses, taste pores, type I cells and type II cells appeared in the taste buds, and fluorescent cells also appeared following treatment of 5-HTP or L-DOPA. During stage 3 (14-21 days), the gustatory cells underwent ultrastructural changes following injection of 5-HTP; i.e. small dense-cored vesicles (30-60 nm) appeared scattered throughout the cytoplasm and were found to intermingle with small clear vesicles accumulated at the presynaptic membranes of afferent synapses, and the electron densities of large dense-cored vesicles (80-100 nm) were elevated as compared with those of untreated mice. Consequently the ability of gustatory cells to take up amine-precursors started simultaneously with the formation of taste pores and mature afferent synapses between the gustatory cells and the sensory nerves.

The ability of amine production by argyrophil cell adenocarcinoma of the endometrium.

The ability of amine production of argyrophil cell adenocarcinoma of the endometrium was studied by wet-histofluorescence method. The intense green and yellow fluorescence was detected for the first time in the cytoplasm of a few tumor cells by injecting amine precursor (L-Dopa) 3mg intraperitoneally to nude mice, who were transplanted serially with argyrophil cell adenocarcinoma of the endometrium. Similar fluorescence was also located in a few epithelial cells of the small intestine prepared from the same nude mice. These findings strongly suggested that these fluorescent products were amines. In order to specify the sorts of amines, the microspectrophotometric analyses are now in progress.

The common peptides and the cytochemistry of their cells of origin.

Thirtyfive biologically active peptides are products of the 40 cells of the APUD series, which constitute the Diffuse Neuroendocrine System (DNES). Twentyone of these peptides are found not only in the cells and processes of the DNES but also in the cells and the processes of the nervous system. Hence the appellation common. To the seven original common cytochemical features of the APUD cells it is possible to add the positive results of 3 subsidiary staining techniques (Lead haematoxylin, Argyrophilia, Formaldehyde-Fluorescamine), a singly cytochemical method (Formaldehyde-ozone), and an immunocytochemical molecular marker method (neuron-specific enolase; NSE). Application of this last method demonstrates NSE in the APUD cells and confirms their origin from "neuroendocrine-programmed epiblast."

Differentiation of Peptococcus and Peptostreptococcus by gas-liquid chromatography of cellular fatty acids and metabolic products.

Gas-liquid chromatographic (GLC) profiles of cellular fatty acids and metabolic products were useful in identifying strains of Peptococcus saccharolyticus, Peptococcus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus micros, and Streptococcus intermedius. The GLC results supported the recent taxonomic decision to transfer aerotolerant Peptostreptococcus species to the genus Streptococcus. Because inconsistencies in the results prevented our differentiating Peptococcus prevotii. Peptococcus magnus, and Peptococcus variabilis by GLC, additional strains will have to been examined. These GLC techniques are amenable to routine use; however, for interlaboratory results to be meaningful, the classification and nomenclature of the anaerobic gram-positive cocci should be standardized.

Identification of Neisseria by electron capture gas-liquid chromatography of metabolites in a chemically defined growth medium.

A dual-purpose study was carried out in an attempt to develop a rapid, sensitive method to identify Neisseria species by gas chromatography and to learn more about the metabolism of these organisms. Sixty-nine isolates of Neisseria were grown in a chemically defined fluid medium; the spent medium was extracted sequentially at pH 2 with diethyl ether and at pH 10 with chloroform. The pH 10 extracts were derivatized with heptafluorobutyric anhydride and analyzed by electron capture gas-liquid chromatography. The resulting spent culture medium electron capture gas-liquid chromatography profiles showed several qualitative and significant quantitative differences among the Neisseria species potentially useful in separating and identifying these organisms. Putrescine and cadaverine which were present in the spent culture medium of some Neisseria, including N. gonorrhoeae, were tentatively identified. Substituting carbohydrates for the chemically defined medium containing glucose in the base medium produced altered profiles with increased quantitative and qualitative differences.