Enzymatic determination of biogenic amines with transglutaminase.

Tyramine, histamine, putrescine and cadaverine, the most common biogenic amines indicating the food quality, were studied in the transglutaminase-catalyzed reaction. Transglutaminase (protein-glutamine gamma-glutamyltransferase EC 2.3.2.13) catalyzes an acyl transfer reaction between a donor substrate and an acceptor substrate (e.g. biogenic amine) and forms a cross-linkage between substrates with a release of ammonia. The reaction can be monitored by measuring the ammonia produced in the reaction. The concentration of produced ammonia was found to be proportional to the concentration of biogenic amine and could hence be used to determination of biogenic amines in food matrixes.

Sterol-induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double-bond position in the sterol tetracyclic ring structure.

We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7-dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 microM cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 nmol.mg-1 protein (from 90 nmol.mg-1 to 190 nmol.mg-1 protein). A similar exposure of cells to different sterol/CyD complexes increased the cell sterol content between 38 and 181 nmol sterol per mg cell protein. In cholesterol-enriched cells, the rate of phosphatidylcholine synthesis was doubled compared to control cells, irrespective of the type of precursor used ([3H]choline, [3H]palmitic acid, or [14C]glycerol). Enrichment of fibroblasts with 7-dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane-bound CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme, was increased by 47 +/- 4% in cholesterol-enriched cells whereas its activity was unchanged in lathosterol-enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down-regulated acetate-incorporation into cholesterol, and upregulated sterol esterification in the sterol-enriched fibroblasts. Using 31P-NMR to measure the lamellar-to-hexagonal (Lalpha-HII) phase transition in multilamellar lipid dispersions, lathosterol-containing membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and either cholesterol or lathosterol (70:30 mol/mol), differential scanning calorimetry also revealed that the Lalpha-HII-transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the Lalpha-HII-transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells.

Proton NMR spectroscopy of the phospholipids in human uterine smooth muscle and placenta.

The parameters regulating the fluidity of myometrial and placental phospholipids include double bonds, fatty acid chain lenght and the cholesterol/phospholipid ratio. The transformation of these parameters was studied during pregnancy and labor. Myometrial and placental tissue samples were collected from 24 patients: 6 were nonpregnant, 6 early-pregnant, 6 late-pregnant not in labor and 6 in labor. After butanol extraction, tissue cholesterol and lipid phosphorus were determined. Proton NMR spectroscopy of the phospholipids was performed at 500 MHz. The myometrial cholesterol/phospholipid ratio was slightly elevated in pregnant patients not in labor. The uterine muscle of the nonpregnant patients contained more CH=CH groups in the phospholipids than that of the late-pregnant patients. There were 29 more double bonds in placental than in uterine tissue per 100 fatty acid molecules. The average fatty acid chain length varied from 14.0 to 18.8. The placenta has longer fatty acid chains than the uterine smooth muscle. The myometrial carbon chain was shortened on the average by 1.4 and the placental by 1.0 carbon atoms, when the patient went into clinical labor. These findings suggest fluidity changes in myometrial and placental phospholipids during human pregnancy and labor.

Structure elucidation of a photodegradation product of ciprofloxacin.

In the photochemical degradation of ciprofloxacin, 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazinyl)-3-quinolone carboxylic acid, two major decomposition products are formed in acidic solution. The main degradation product, after both artificial and daylight exposure, was 7-amino-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolone carboxylic acid. This product was also the dominating compound after more than 5 h irradiation with a high-pressure mercury lamp in aqueous solutions at pH < or = 2 when the solvent additionally contained water-miscible organic solvent. The structure of the isolated compound was elucidated on the basis of the chemical behaviour in thin-layer and high-performance liquid chromatography, and of information from infrared, ultraviolet, mass and nuclear magnetic resonance spectra.

Isolation and structure elucidation of an intermediate in the photodegradation of ciprofloxacin.

Ciprofloxacin decomposes photochemically in aqueous solutions at acidic pH forming two major degradation products. One of the products, isolated from irradiated solutions by flash chromatography, was 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-3-quinoline carboxylic acid. The compound was an intermediate in the photochemical process, which degraded after longer exposure with a high-pressure mercury lamp to an aromatic amino-compound, 7-amino-1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-3-quinoline carboxylic acid. The structure of the intermediate was elucidated on the basis of information from ultraviolet, mass and nuclear magnetic resonance spectra.

Tissue phospholipids during human pregnancy by 31P NMR: myometrium, decidua, placenta and fetal membranes.

The biophysical environment formed by phospholipids, rather than the amount of functional proteins, can be rate limiting for factors controlling myometrial contractility and pregnancy maintenance. We therefore studied myometrial, decidual, placental and fetal membrane phospholipids using the 31P NMR spectrum. This enabled us to identify bulk phospholipids over 0.05 mmol/kg. The method was checked for reliability for the reproductive tissues studied. The chemical shift of phospholipid standards was slightly different according to whether a single compound or a mixture was analyzed. The bulk phospholipids found were phosphatidylcholine (PC), phosphatidylethanolamine, sphingomyelin (SM) and phosphatidylinositol. The ratio PC/SM decreased during pregnancy in the decidua, placenta and fetal membranes, but not in the myometrium. Pregnancy did not induce significant changes in the total myometrial phospholipids. Their composition was stable even during clinical labor. The fetal tissues, placenta and fetal membranes contained about twice as much phospholipid as the maternal tissues, myometrium and decidua. There was no sign of lysocompounds, cardiolipin or phosphatidic acid. This supports the view that the extraction and analyzing techniques used earlier probably created artefacts. The increased fluidity of the myometrial and placental phospholipids during pregnancy may depend on factors other than the composition of phospholipids.

Phosphorus-31 nuclear magnetic resonance of aspartate aminotransferase from chicken heart cytosol.

31P-nuclear magnetic resonance and absorption spectra of cytosolic chicken aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been recorded in the pH range from 5 to 8.5. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; the chemical shift change was 0.35 ppm. The pK value found by spectrophotometric titration of the enzyme proved to be about 6.0. The monoanion-dianion transition of the 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in the 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein-bound coenzyme is in a dianionic form throughout the investigated pH range; the pH-dependence of the 31P chemical shift may be due to a conformational change at the active site. In the presence of 100 mM succinate, 6 mM aminooxyacetate or 25 mM cycloserine, the 31P chemical shift is insensitive to pH variations.

[31P-NMR spectra of aspartate aminotransferase from cytosol of the chicken heart]. / Issledovanie spektrov 31P-IaMR aspartat-aminotransferazy iz tsitozolia kurinykh serdets.

31P NMR spectra of the cytosolic chicken aspartate aminotransferase have been recorded at 161.7 MHz in the pH range of 5.7 to 8.2. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; difference in the chemical shift at pH 5.7 and 8.2 is only 0.35 ppm. The monoanion-dianion transition of 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein--bound coenzyme is in dianionic form throughout the investigated pH range; the small pH-dependent change of chemical shift may be due to a protein conformational change that affects O-P-O bond angle. In the presence of the 0.1 M succinate, 31P chemical shift of the enzyme remains constant in the pH range of 5.0 to 8.3.