1,4-Thiomorpholine-3,5-dicarboxylic acid, a novel cyclic imino acid detected in bovine brain

Gas-liquid chromatography of enriched bovine brain extract revealed the occurrence of several sulfur-containing compounds. By co-chromatography with authentic product and by mass-spectrometric analysis, one of these compounds has been identified as 1,4-thiomorpholine-3,5-dicarboxylic acid (TMDA). The possible derivation of TMDA from lanthionine is discussed. This represents the second S-containing cyclic amino acid so far discovered in a mammalian brain whose physiological significance has not yet been explored.     

Isolation and sequence of a cDNA clone that contains the entire coding region for chicken smooth-muscle alpha-tropomyosin

We have constructed a cDNA-expression library of approximately 100,000 members from embryonic chicken smooth-muscle mRNA using the plasmid-expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, we have identified and isolated clones encoding smooth-muscle tropomyosin. Plasmid pSMT-10 (approximately 1100 base pairs) was found to hybrid-select mRNA for smooth-muscle alpha-tropomyosin. DNA-sequence analysis revealed that pSMT-10 contained the entire coding region for alpha-tropomyosin and portions of the 5'- and 3'-untranslated regions. Comparison of the derived amino acid sequence of smooth-muscle alpha-tropomyosin with known skeletal-muscle (rabbit and chicken) and platelet (equine) sequences revealed extensive homology between the various proteins. The smooth-muscle tropomyosin shows the greatest sequence divergence from the skeletal-muscle tropomyosins at the COOH-terminal region. In contrast, the smooth-muscle tropomyosin is most homologous to the platelet tropomyosin at the COOH-terminal end. The relationship of the various tropomyosin sequences to function (e.g. interactions with troponin) are considered.     

Interaction between 1,4-thiazine derivatives and D-amino-acid oxidase

Aminoethylcysteine-ketimine (2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid) strongly inhibits D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3). The inhibition is purely competitive (Ki = 3.3 X 10(-7) M). Aminoethylcysteine-ketimine modifies the visible spectrum of the enzyme: the absorption maxima of bound FAD shift from 375-455 nm to 385-445 nm with a definite shoulder at 465 nm; the appearance of a large absorption band centered at 750 nm may be due to a charge-transfer complex formation. The dissociation constant for the aminoethylcysteine-ketimine-enzyme complex, calculated by a photometric procedure (4 X 10(-7) M), is in good agreement with kinetic data. The dicarboxylic analogue of this inhibitor (lanthionine-ketimine) is ineffective in D-amino-acid oxidase inhibition and does not produce any spectral modification of the enzyme. These results confirm structural requirements for D-amino-acid oxidase inhibitor reported by other researchers. Ketimine reduced forms (thiomorpholine-2-carboxylic acid and thiomorpholine-2,6-dicarboxylic acid) are chemically synthesized and checked as D-amino-acid oxidase substrates: only thiomorpholine-2-carboxylic acid is oxidized to aminoethylcysteine-ketimine (Km = 2 X 10(-4) M).     

The Formation of Otoliths in the Frog Rana esculenta. Scanning Electron Microscopic and X-Ray Diffraction Studies

Two crystal forms of calcium carbonate were observed: calcite (utricle) and aragonite (saccule, lagena, endolymphatic sac). The first step in otolith formation is the appearance of organic structures in the macula. The subsequent step is characterized by fast growing primitive crystals with a prismatic habitus that successively transform into adult or mature crystals. With the metamorphosis, the aragonite crystals of the endolymphatic organ show clear signs of erosion that can be related to a process of CaCO₃ mobilization from such deposits.     

Similarity of the oxidation products of L-cystathionine by L-amino acid oxidase to those excreted by cystathioninuric patients

L-Cystathionine is oxidized by snake venom L-amino acid oxidase at a rate about half that with L-leucine at pH 8.5. The appearance of an absorbance at 296 nm and quantitation of the products of oxidation in the presence of catalase indicate formation in the solutions of a seven-membered ketimine ring produced by cyclization of the monoamino monoketo derivative of cystathionine. A limited double deamination has also been observed. In the absence of catalase, S-(carboxymethyl)homocysteine and S-(beta-carboxyethyl)cysteine have been identified together with ninhydrin-unreactive compounds yielding the above mentioned carboxy compounds upon hydrolysis with HCl. Authentic samples of the monoamino monoketo analogs of cystathionine have been prepared and compared with the enzymatic products. Cyclization of the synthetic products into the ketimine ring is pH-dependent as established by UV spectrum and other assays. Compounds derived from either the oxidation or the reduction of the ketimine have been prepared. It was found that many products of enzymatic and chemical changes of cystathionine and its ketimine described in the present paper are identical with those identified in the urine of cystathioninuric patients. This result indicates the occurrence in humans of secondary metabolic routes of cystathionine centered on the production of cystathionine ketimine, in equilibrium with the open form, which in cystathioninurics is revealed by the lack of cystathionase.     

Effect of carbon dioxide on differentiation and on the level of a soluble b-type cytochrome in Phycomyces blakesleeanus

A soluble b-type cytochrome has been detected and partly characterized in mycelial extracts of Phycomyces blakesleeanus. As it is already known, CO₂ delays sporangiophorogenesis, but it also lowers the level of this cytochrome. A possible causal relationship between sporangiophorogenesis and the b-type-cytochrome level may exist. There is some correlation between the extent of the delay of sporangiophorogenesis and of the decrease in cytochrome-b level in wild type and mutants that are either resistant or sensitive to CO₂.