Transaminases in the acquired pellicle.

OBJECTIVE: Transaminases (AST, aspartate amino transferase; ALT, alanine amino transferase) are relevant enzymes in physiology and pathology of the human organism. The aim of the present in situ study was to demonstrate the presence of these enzymes in the enamel pellicle. METHODS: Bovine enamel slabs were fixed on buccal sites of individual upper jaw splints and worn for 3, 30 and 120 min by 5 subjects to allow pellicle formation. The in situ pellicles were tested for AST and ALT. Enzyme activities were measured photometrically via determination of the products pyruvate and oxalacetate using lactate-dehydrogenase and malate-dehydrogenase, respectively. RESULTS: Enzymatic AST- as well as ALT-activities are present in the acquired pellicle within 3 min. The enzyme activities exposed at the pellicles' surfaces increased slightly with the pellicle formation time (ANOVA, AST: n.s., ALT: p=0.021). However, the two enzymes show considerable intraindividual and interindividual variability. The mean AST-activity of the pellicle amounted to 1.07+/-0.81 mU/cm(2) (ALT 1.18+/-0.52 mU/cm(2)). The ALT-activity of the centrifuged saliva was 26.62+/-11.09 mU/ml (AST 35.98+/-29.35 mU/ml). CONCLUSIONS: AST as well as ALT are present in the in situ pellicle layer and may contribute to the intrinsic maturation of pellicle proteins.

(S)-(-)-alpha,alpha-Di(2-naphthyl)-2-pyrrolidinemethanol, a useful tool to study the recognition mechanism in chiral ligand-exchange chromatography.

A dynamic coating of the RP-18 carbon chain layers with the new chiral selector (S)-(-)-alpha,alpha-di(2-naphthyl)-2-pyrrolidinemethanol allowed the formation of a mixed chiral stationary phase that has been used in the separation of a selected set of amino acid racemates. Both a representative model and classification structure-property relationship studies have been performed in order to study the contribution of hydrophobic, bulky and electron-donating groups in the side chain of the chiral selector to the mechanism of chiral recognition.

Aspartate dehydrogenase in vitamin B12-producing Klebsiella pneumoniae IFO 13541.

We found a new reaction of aspartic acid dehydrogenation, catalyzed by NADP(+)-dependent aspartate dehydrogenase, in vitamin B12-producing Klebsiella pneumoniae IFO 13541. The enzyme, which was purified from a crude extract of K.pneumoniae IFO 13541, catalyzes the oxidative deamination of aspartic acid to form oxaloacetic acid. This enzyme had a molecular mass of about 124 kDa consisting of two identical subunits. L-Aspartic acid was a substrate, although D-aspartic acid and L-glutamic acid were inactive. The enzyme showed maximal activity at about pH 7.0-8.0 for the oxidative deamination of L-aspartic acid, and it required NADP+ as a coenzyme, while NAD+ was inactive.

Effect of emeriamine, an inhibitor of fatty acid oxidation, on metabolic fate of a geometrical isomer of linoleic acid in perfused rat liver.

To estimate the relative significance of exogenous vs. endogenous fatty acids in increasing hepatic triacylglycerol secretion following an inhibition of fatty acid oxidation by emeriamine, livers from 2-d-fasting rats were perfused with or without an inhibitor in the presence of a geometrical isomer of linoleate (linolelaidic acid, trans,trans-9,12-octadecadienoic acid). Emeriamine added to the perfusion medium at 2 h of the recirculating perfusion period caused immediate and complete cessation of ketone body production while it increased triacylglycerol and cholesterol secretion by the liver without affecting uptake of exogenous linolelaidic acid. The increase in the triacylglycerol secretion by emeriamine was accompanied by a marked increase in the proportion of linolelaidic acid in this lipid molecule in the perfusate and in the liver. The calculated amounts of exogenous linolelaidate, compared with those of endogenous fatty acids in the secretory triacylglycerol, suggested that the former compared with the latter contributes more to the drug-mediated increase in triacylglycerol secretion. This drug caused a marked reduction of mitochondrial carnitine palmitoyltransferase activity in perfused liver. These results suggest that a blockade of fatty acid oxidation by emeriamine, through an inhibition of carnitine palmitoyltransferase, diverts predominantly the exogenous free fatty acids from oxidation to the esterification pathway and subsequently stimulates the synthesis and secretion of triacylglycerol-rich lipoproteins.

Assay of blood and tissue oxaloacetate and alpha-ketoglutarate by isotope dilution gas chromatography-mass spectrometry.

The assay of oxaloacetate and alpha-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological concentrations of these metabolites by isotope dilution gas chromatography-mass spectrometry. Samples are spiked with the corresponding internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the [M-57]+ ion in electron impact. Although the internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting [1,2,3,6-13C4]citrate with citrate lyase, and L-[U-13C5]glutamate with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of [1,2,3,6-13C4]citrate is prepared from L-[U-13C4]aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and alpha-ketoglutarate in human blood and rat liver.

A sensitive multienzymatic assay for the measurement of pyruvate, dihydroxyacetone phosphate, oxaloacetate, and acetoacetate in clear extracts from biological samples.

A multienzymatic method for the measurement of pyruvate, dihydroxyacetone phosphate, oxaloacetate, and acetoacetate is presented. The determination procedure is considered suitable because it is simple, sensitive, and its advantages could be demonstrated by comparison with the original methods.

[The role of ketone bodies in energy supply in cold-blooded animals during winter starvation]. / Rol' ketonovykh tel v énergoobespechenii poikilotermnykh organizmov v usloviiakh zimnego golodaniia.

Natural winter starvation has been studied for its effect on the content of ketone bodies, oxaloacetate, glucose, 3-oxybutyrate-dehydrogenase activity level in the carp fry tissue. A compensatory mechanism of the energy supply in peripheral tissues is found proceeding by formation of ketone bodies in the liver and their distribution in the tissues of white muscles and brain. For the latter the ketone bodies in wintering serve as an additional oxidation substrate.

Fibre-optic biosensor based on luminescence and immobilized enzymes: microdetermination of sorbitol, ethanol and oxaloacetate.

We have investigated highly selective and ultrasensitive biosensors based on luminescent enzyme systems linked to optical transducers. A fibre-optic sensor with immobilized enzymes was designed; the solid-phase bioreagent was maintained in close contact contact with the tip of a glass fibre bundle connected to the photomultiplier tube of a luminometer. A bacterial luminescence fibre-optic sensor was used for the microdetermination of NADH. Various NAD(P)-dependent enzymes, sorbitol dehydrogenase, alcohol dehydrogenase and malate dehydrogenase, were co-immobilized on preactivated polyamide membranes with the bacterial system and used for the microdetermination of sorbitol, ethanol and oxaloacetate at the nanomolar level with a good precision.

Flow-injection analysis of amino acids and their metabolites by immobilized vitamin B6-dependent enzymes. Sensitive determination of L-aspartate, L-glutamate, 2-oxoglutarate, and oxaloacetate.

Sensitive flow-injection analyses of aspartate, glutamate, 2-oxoglutarate, and oxaloacetate were developed. The analytes were enzymatically coupled with NADH which was monitored by light emission from immobilized bacterial bioluminescence enzymes. Aspartate (or oxaloacetate) was assayed on the basis of NADH consumption by introducing the sample through a coimmobilized aspartate aminotransferase-malate dehydrogenase column. The assay responded linearly from 100 pmoles to 5 nmoles per assay. Glutamate (2-oxoglutarate) was determined by formation of NADH in the glutamate dehydrogenase reaction. The measuring range for glutamate was from 10 pmoles to 100 nmoles per assay. The precision of the flow-injection method was generally excellent, and the sensitivities of the described assays were 100-1000-fold higher than with spectrophotometric methods. The immobilized enzyme preparations were stable for several months in storage, and the enzyme columns could be used for 600-800 analyses. Flow-injection analyses of amino acids and related compounds by NADH/bioluminescence-coupled reactions provide a sensitive, fast, and inexpensive assay method for a wide variety of purposes.

Fluorometric determination of alpha-ketosuccinamic acid in rat tissues.

A method for the fluorometric determination of alpha-ketosuccinamic acid, the alpha-keto acid analog of asparagine, is described. The procedure involves the hydrolysis of alpha-ketosuccinamate to oxaloacetate by omega-amidase followed by NADH-dependent reduction of oxaloacetate to malate by malate dehydrogenase. A correction for endogenous oxaloacetate is made by using control samples lacking omega-amidase. Of the rat tissues investigated, liver contained the highest concentration, followed by kidney (53 +/- 6 (n = 11) and 18 +/- 3 (n = 3) mumol/kg wet wt, respectively). alpha-Ketosuccinamate was not detected in brain (less than 8 mumol/kg wet wt). Some chemical properties of alpha-ketosuccinamate were investigated. Concentrated solutions of sodium alpha-ketosuccinamate frozen for extended periods and the solid sodium salt of alpha-ketosuccinamate dimer heated to 130 degrees C are converted to at least 10 products by processes involving dimerization, dehydration, and decarboxylation. Isobutane chemical ionization mass spectral analysis (170-230 degrees C) of the free acid monomer yielded similar products. Many of the breakdown products were identified as di- and monoheterocyclic compounds, some of which are known to be of biological importance.

Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats.

In isolated hepatocytes from normal fed rats, the subcellular distribution of malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH has been determined by a modified digitonin method. Incubation with various substrates (lactate, pyruvate, alanine, oleate, oleate plus lactate, ethanol and aspartate) markedly changed the total cellular amounts of metabolites, but their distribution between the cytosolic and mitochondrial compartments was kept fairly constant. In the presence of lactate, pyruvate or alanine, about 90% of cellular aspartate, malate and oxaloacetate, and 50% of citrate was located in the cytosol. The changes in acetyl-CoA in the cytosol were opposite to those in the mitochondrial space, the sum of both remaining nearly constant. The mitochondrial acetyl-CoA/CoASH ratio ranged from 0.3-0.9 and was positively correlated with the rate of ketone body formation. The mitochondrial/cytosolic (m/c) concentration gradients for malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH averaged from hepatocytes under different substrate conditions were determined to be 1.0, 8.8, 1.6, 2.2, 0.5, 0.7, 13 and 40, respectively. From the distribution of citrate, a pH difference of 0.3 across the inner mitochondrial membrane was calculated, yet lower values resulted from the m/c gradients of 2-oxoglutarate, glutamate and malate. The mass action ratios for citrate synthase and mitochondrial aspartate aminotransferase have been calculated from the metabolite concentrations measured in the mitochondrial pellet fraction. A comparison with the respective equilibrium constants indicates that in intact hepatocytes, neither enzyme maintains its reactants at equilibrium. On the assumption that mitochondrial malate dehydrogenase and 3-hydroxybutyrate dehydrogenase operate near equilibrium, the concentration of free oxaloacetate appears to be 0.3-2 micron, depending on the substrate used. Plotting the calculated free mitochondrial oxaloacetate concentration against the citrate concentration measured in the mitochondrial pellet yielded a hyperbolic saturation curve, from which an apparent Km of citrate synthase for oxaloacetate in the intact cells of 2 micron can be derived, which is comparable to the value determined with purified rat liver citrate synthase. The results are discussed with respect to the supply of substrates and effectors of anion carriers and of key enzymes of the tricarboxylic acid cycle and fatty acid biosynthesis.

[Characteristics of the activity of "satellite" enzymes of gluconeogenesis in the oocytes and embryos of loach]. / Osobennosti aktivnosti "satellitnykh" fermentov gliukoneogeneza v ootistakh i zarodyshakh v'iuna

The activity of "satellite" enzymes related to gluconeogenesis has been measured in the oocytes and embryos at the early stages of loach (Misgurnus fossilis L.) embryogenesis. The activity of pyruvate dehydrogenase increase during oocyte maturation by 30%, remains constant at the cleavage and blastula stages and decreased on the onset of gastrulation. In the both oocytes and embryos pyruvate dehydrogenase has been found only in the active form. The activity of citrate synthase, malate dehydrogenase and pyruvate carboxylase remained constant during oocyte maturation and et all early stage of embrional development. Citrate lyase and "malic"-enzyme were not found, Oocyte maturation is followed by a considerable increase in the malate and oxalacetate content, the level of pyruvate and acetyl-CoA being found invariable.

Colorimetric enzymatic determination of serum total carbon dioxide, as applied to the Vickers Multichannel 300 discrete analyzer.

Phosphoenolpyruvate carbosylase (EC 4.1.1.31) has been used in developing a simple, inexpensive colorimetric assay for serum total carbon dioxide in an open system. The oxaloacetate formed by the action of the enzyme on bicarbonate and phosphoenolpyruvate is measured by use of the diazonium salt of Fast Violet B. Interferences from billirubin, pyruvate, and drugs are negligible. Acetoacetate interference is significant only in highly ketotic samples, and a serum blank corrects for it. Serum protein interference is equivalent to 3.3 plus or minus 1.25 mmol of CO2 per liter and hence is sufficiently constant to be corrected for by use of a serum standard or serum blank. The method has been applied to the Vickers M-300 and D-300 systems and within-batch standard deviations of plus or minus 0.1 to plus or minus 0.6 mmol/liter have been observed. Excellent correlation with orthodox techniques has been obtained.