Intracellular localization of GDP-l-fucose: Glycoprotein and CMP-sialic acid: Apolipoprotein glycosyltransferases in rat and pork livers

A GDP-l-fucose:glycoprotein fucosyltransferase which transfers l-fucose to terminal β-N-acetyl-d-glucosaminyl residues of sialidase-, β-galactosidase-treated α₁-acid glycoprotein and a CMP-sialic acid:glycoprotein sialyltransferase acting on sialidase-treated apolipoprotein-Ala₁ from human very low density lipoprotein have been shown to be concentrated in rat liver Golgi apparatus preparations at enrichments of 40- and 45-fold, respectively, and in pork liver Golgi-rich fractions at enrichments of 35- and 20-fold, respectively. A second fucosyltransferase acting on sialidase-treated α₁-acid glycopretein was absent from rat liver and was enriched only 13-fold in a pork liver Golgi-rich fraction. The smooth-surfaced microsome fraction was the only other rat liver subcellular fraction with appreciable levels of the GDP-l-fucose: β-N-acetyl-d-glucosaminide fucosyltransferase and the lipoprotein sialyltransferase (enrichments of 2.6- and 5.2-fold, respectivley). This enrichment could not be attributed to the plasma membrane content of the smooth microsome fraction since plasma membrane fractions from rat liver were shown to have relatively low concentrations of these two transferases (enrichments of 0.3 or less). Rat liver plasma membrane was also shown to have similarly low relative specific activities for three other glycosyltransferases (sialyl-, galactosyl-, and N-acetylglucosaminyl-). The accurate determination of the glycosyltransferase activities of the plasma membrane fraction required the use of relatively low concentrations of plasma membrane and relatively high concentrations of nucleotide-sugars in order to avoid interference by the high nucleotide-sugar pyrophosphatase and hydrolase activities of this fraction.     

The effect of acetaldehyde on gluconeogenesis from xylitol, sorbitol, and fructose by isolated rat liver cells.

In isolated rat liver cells, ethanol inhibited gluconeogenesis from xylitol and sorbitol but not from fructose. Acetaldehyde, at initial concentrations of 0.2, 0.5, and 1.0 mm, stimulated gluconeogenesis from xylitol and sorbitol in the absence of pyrazole but inhibited in the presence of pyrazole. There was no effect with fructose. Acetate had no effect. Methylene blue and pyruvate (but not lactate) prevented the stimulatory as well as the inhibitory effects of acetaldehyde. Acetoacetate (but not β3-hydroxybutyrate) prevented, to a large extent, the inhibitory effects of low (but not high) concentrations of acetaldehyde. The inhibition by low concentrations of acetaldehyde appears to be mediated via acetaldehyde oxidation in the mitochondria, whereas the inhibition by high concentrations of acetaldehyde appears to reflect acetaldehyde oxidation in the cytosol. These data indicate that the inhibitory action of ethanol on glucose production from xylitol and sorbitol can be reproduced by physiological concentrations of acetaldehyde. Changes in the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio produced during acetaldehyde metabolism appear to be responsible for these effects of acetaldehyde. These changes may contribute to the actions of ethanol on gluconeogenesis from these substrates.     

Amidino-substituted aromatic heterocycles as probes of the specificity pocket of trypsin-like proteases.

The effect of pargyline on the uptake of acetaldehyde (in the presence of pyrazole) by isolated rat liver cells was studied after incubating the liver cells for 0, 10, 30, 45, and 60 min with 0.40, 1.30, and 2.6 mm pargyline. Without any incubation period, pargyline had no effect on acetaldehyde uptake. With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. This suggests the possibility that pargyline is metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action. Pargyline was also a more effective inhibitor of the uptake of lower concentrations of acetaldehyde, e.g., 0.167 mm, than of higher concentrations (1.0 mm) of acetaldehyde, especially after short incubation periods or when pyrazole was omitted from the reaction medium. After a 20- to 30-min incubation period, pargyline inhibited the control rate of ethanol oxidation by the liver cells, as well as the accelerated rate of ethanol oxidation found in the presence of pyruvate or an uncoupling agent. Pargyline had no effect on hepatic oxygen consumption. During ethanol oxidation, a time-dependent release of acetaldehyde into the medium was observed. Pyruvate, by increasing the rate of ethanol oxidation, increased the output of acetaldehyde five- to tenfold. Pargyline increased the output of acetaldehyde two- to threefold, despite decreasing the rate of ethanol metabolism by the liver cells. These data indicate that pargyline inhibits the low K_m aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. Although most of the acetaldehyde generated during the oxidation of ethanol is removed by the liver cells in an effective manner, changes in the activity of aldehyde dehydrogenase or the rate of acetaldehyde generation significantly alter the hepatic output of acetaldehyde.     

Destruction of highly purified cytochromes P-450 associated with metabolism of fluorinated ether anesthetics

The effects of ethanol and acetaldehyde on rat intestinal microvillus membrane integrity and glucose transport function were examined in vitro with purified membrane vesicles. Ethanol could influence glucose transport function by alterations in the conformation of the carrier, the lipid environment surrounding the carrier, or in the transport driving force (Na⁺ electrochemical gradient). Due to the rapid nature of glucose uptake, transport was assayed with the use of an apparatus that permitted uptake measurements as early as 1 s. Ethanol (340 mm) partially and acetaldehyde (44 mm) completely inhibited concentrative glucose uptake throughout the 1-min time course. Their inhibitory effects were reversible and irreversible, respectively. Kinetic measurements made during the initial rate of uptake (at 2 s) with various concentrations of glucose (0.05–8 mm) showed that ethanol and acetaldehyde both caused a decrease in V. Although ethanol did not substantially alter the transport K_m, acetaldehyde increased the K_m almost 50%. To determine whether ethanol or acetaldehyde directly interfered with glucose carrier function, uptake was measured in the presence of equilibrated Na⁺. Only acetaldehyde had a significant inhibitory effect under these conditions. Membrane permeability, as determined by efflux of preloaded 6-carboxyfluorescein dye, increased upon exposure of the vesicles to ethanol or acetaldehyde. Membrane fluidity measurements by fluorescence polarization showed that only acetaldehyde had a significant fluidizing effect. These results indicate that ethanol and acetaldehyde acted to perturb membrane integrity and inhibited glucose uptake indirectly by allowing the Na⁺ gradient to dissipate. Acetaldehyde, which had a stronger inhibitory effect than ethanol, appeared also to directly inhibit carrier function.     

Localization and characteristics of rat liver mitochondrial aldehyde dehydrogenases.

Two NAD-dependent aldehyde dehydrogenase enzymes from rat liver mitochondria have been partially purified and characterized. One enzyme (enzyme I) has molecular weight of 320,000 and has a broad substrate specificity which includes formaldehyde; NADP is not a cofactor for this enzyme. This enzyme has K_m values for most aldehydes in the micromolar range. The isoelectric point was found to be 6.06. A second enzyme (enzyme II) has a molecular weight of 67,000, a K_m value for most aldehydes in the millimolar range but no activity toward formaldehyde. NADP does serve as a coenzyme, however. The isoelectric point is 6.64 for this enzyme. By utilization of the different substrate properties of these two enzymes it was possible to demonstrate a time-dependent release from digitonin-treated liver mitochondria. The high K_m, low molecular weight enzyme (enzyme II) is apparently in the intermembrane space while the low K_m, high molecular weight enzyme (enzyme I) is in the mitochondrial matrix and is most likely responsible for oxidation of acetaldehyde formed from ethanol.     

Cyclic-voltammetric determination of cysteine in rat organs after intravenous injection

A method of cyclic voltammetry was used for the determination of cysteine in rat organs in vivo after its intravenous injection. The cyclic polarization enabled a periodic reactivation of the platinum test electrode surface so that reproducible results could be obtained. The peaks of the I-E curve which were due to oxidation of cysteine depended for reproducibility on the concentration of cysteine injected. We attempted to use the method described for the assessment of kidney tissue blood supply before transplantation.     

Acetaldehyde kinetics of enological yeast during alcoholic fermentation in grape must

Acetaldehyde strongly binds to the wine preservative SO₂ and, on average, causes 50–70 mg l^?1 of bound SO₂ in red and white wines, respectively. Therefore, a reduction of bound and total SO₂ concentrations necessitates knowledge of the factors that affect final acetaldehyde concentrations in wines. This study provides a comprehensive analysis of the acetaldehyde production and degradation kinetics of 26 yeast strains of oenological relevance during alcoholic fermentation in must under controlled anaerobic conditions. Saccharomyces cerevisiae and non-Saccharomyces strains displayed similar metabolic kinetics where acetaldehyde reached an initial peak value at the beginning of fermentations followed by partial reutilization. Quantitatively, the range of values obtained for non-Saccharomyces strains greatly exceeded the variability among the S. cerevisiae strains tested. Non-Saccharomyces strains of the species C. vini, H. anomala, H. uvarum, and M. pulcherrima led to low acetaldehyde residues (<10 mg l^?1), while C. stellata, Z. bailii, and, especially, a S. pombe strain led to large residues (24–48 mg l^?1). Acetaldehyde residues in S. cerevisiae cultures were intermediate and less dispersed (14–34 mg l^?1). Addition of SO₂ to Chardonnay must triggered significant increases in acetaldehyde formation and residual acetaldehyde. On average, 0.33 mg of residual acetaldehyde remained per mg of SO₂ added to must, corresponding to an increase of 0.47 mg of bound SO₂ per mg of SO₂ added. This research demonstrates that certain non-Saccharomyces strains display acetaldehyde kinetics that would be suitable to reduce residual acetaldehyde, and hence, bound-SO₂ levels in grape wines. The acetaldehyde formation potential may be included as strain selection argument in view of reducing preservative SO₂ concentrations.     

Evaluation of a role of acetaldehyde in the mechanism of inhibition of p-nitroanisole O-demethylation in isolated hepatocytes by ethanol

The rate of p-nitroanisole O-demethylation is markedly inhibited by ethanol. To evaluate a role of acetaldehyde in the inhibition by ethanol, a comparison was made of the effects of ethanol and acetaldehyde on the metabolism of p-nitroanisole by isolated liver cells. No effect on the metabolism of p-nitroanisole was found at low concentrations of acetaldehyde (<0.5 mm), whereas inhibition occurred at high concentrations (1 mm). In fact, acetaldehyde was not any more inhibitory than crotonaldehyde, which is a poor substrate for the low-K_m mitochondrial aldehyde dehydrogenase. Cyanamide, an inhibitor of acetaldehyde oxidation, did not prevent the inhibition by ethanol. Crotonol, an alcohol that does not change the mitochondrial redox state, in contrast to ethanol, proved to be a more effective inhibitor of the metabolism of p-nitroanisole than ethanol. Greater sensitivity to crotonol was also found in isolated microsomes and may reflect hydrophobic effects by crotonol, relative to ethanol. These results suggest that although high levels of acetaldehyde can be inhibitory, physiological levels of acetaldehyde did not affect the metabolism of p-nitroanisole. It is unlikely that acetaldehyde itself plays a major role in the mechanism by which ethanol inhibits the metabolism of p-nitroanisole. The inhibition of p-nitroanisole O-demethylation by ethanol was prevented by pyruvate or fructose, but not by xylitol, sorbitol, or lactate. All these substrates by themselves stimulated metabolism of p-nitroanisole. Pyruvate and glyceraldehyde (which arises from the metabolism of fructose) can oxidize cytosolic NADH. These results suggest that the generation of cytosolic NADH from the oxidation of ethanol, the subsequent requirement for substrate shuttles to transfer NADH into the mitochondria, and redox inhibition of the citric acid cycle, interfere with the transport of NADPH out of the mitochondria, and consequently with drug metabolism.     

Palmitate oxidation by rat skeletal muscle mitochondria: Comparison of polarographic and radiochemical experiments

Oxidation of palmitate by rat skeletal muscle mitochondria was determined polarographically and radiochemically under state 3 conditions. Maximal oxidation rate is reached at 4 μm palmitate, palmitoyl-CoA, or palmitoyl-l-carnitine. At palmitoyl-CoA concentrations higher than 30 μm oxidation is inhibited. At limiting substrate concentrations as used in polarographic experiments palmitate is totally degraded to CO₂. At higher concentrations the palmitate molecule is only partially degraded, due to the accumulation of intermediates. Citric acid cycle intermediates, especially 2-oxoglutarate, accumulate during oxidation of palmitate in the presence of malate. It is suggested that this accumulation is stimulated by dicarboxylate exchange. The rate of formation of ¹⁴CO₂ and ¹⁴C-labeled perchloric acid-soluble products is higher from [1-¹⁴C]palmitate than that from [U-¹⁴C]palmitate. This difference, which is enhanced by higher carnitine concentrations indicates incomplete oxidation during the β-oxidation in state 3. The simultaneous determination of ¹⁴CO₂ production and ¹⁴C-labeled perchloric acid-soluble products appears to be a more accurate and sensitive method for measuring ¹⁴C-fatty acid oxidation than that of ¹⁴CO₂ production alone.     

A note on osmotic pressure measured with ionophore treated red blood cells

A method is described by which the osmotic pressure of macromolecules or many low molecular weight substances can be measured relative to the known osmotic pressure of a reference substance. Measurements can also be made in the presence of univalent electrolytes. The method involves the use of ionophore treated mammalian red blood cells as osmometers. Details are given for the establishment of the isosmotic identity line for dextran M_w = 9 400, M_n = 5 500 and sucrose using nystatin treated human red blood cells. The sucrose concentrations used were from 20 to 33 mOsm (50–80 kPa).     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

Two-dimensional electrophoresis of membrane proteins factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI > 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112–120) gives good resolution of proetins of pI 5–8, including cytochromes P-450, but fails to resolve a major acidic protein of pI < 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R·N⁺(CH₃)₂·CH₂CH₂CH₂SO₃^? are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C₁₄ or C₁₆) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

A sensitive assay for the determination of propionyl-CoA in biological material

A method has been developed for the determination of low amounts of propionyl-CoA in biological material. The method involves ¹⁴CO₂ fixation by propionyl-CoA in the presence of purified propionyl-CoA carboxylase. Values for propionyl-CoA in rat liver in vivo and in isolated rat livers perfused in the presence of propionate are reported.     

Acetaldehyde binding increases the catabolism of rat serum low-density lipoproteins

Acetaldehyde was found to form adducts with rat serum lipoproteins. The binding of [14C]acetaldehyde to lipoproteins was studied at low concentrations which are known to exist during ethanol oxidation. The amount of lipoprotein adducts was a linear function of acetaldehyde concentration up to 250 microM. Incubation of rat plasma low-density lipoproteins (LDL) with 200 microM acetaldehyde increased the disappearance rate of the 3H-label from the cholesterol ester moiety of LDL injected into normal rats. The data show that even low concentrations of acetaldehyde are capable of affecting LDL metabolism. These findings may provide an explanation for the low concentrations of serum LDL in alcoholics.     

Effect of melittin-induced membrane alterations on rat heart adenylate cyclase activity

Melittin, a surface-active, 26-amino acid polypeptide from bee venom, has been reported to alter a variety of membrane properties including stability, permeability, and fluidity, the latter having been shown to be altered in a biphasic manner. Melittin induced a biphasic alteration of rat heart microsomal adenylate cyclase activity, stimulating it at low concentration (<30 μg/ml) and inhibiting it at higher concentrations (100 μg/ml or higher). Melittin potentiated sodium fluoride and 5′-guanylylimidodiphosphate activation of adenylate cyclase below 40 μg/ml but it inhibited at high concentrations, except in the presence of high concentrations of 5′-guanylylimidodiphosphate (10^?4m). Basal and fluoride-activated adenylate cyclase exhibited no significant change in the K_m for ATP in the presence of melittin at <40 μg/ml, but the V was elevated. Potentiation by melittin of adenylate cyclase was observed at all fluoride, 5′-guanylylimidodiphosphate, and magnesium concentrations tested. The observed effects of melittin on rat heart adenylate cyclase are consistent with it acting by altering the properties of membrane lipids with which the enzyme is associated.     

Determination of acetaldehyde in biological samples by gas chromatography with electron-capture detection

A simple specific assay was developed for the determination of acetaldehyde in biological samples. Acetaldehyde was derivatized to 2,4-dinitrophenylhydrazone, which was determined by gas chromatography with electron-capture detection. The use of this detection method is an important device to which no one drew notice. This procedure was very simple and so sensitive that as little as 500 fmol of acetaldehyde could be measured in aqueous solution. The calibration curve of acetaldehyde was linear at least up to 40 μM. Its recoveries from human plasma and rat liver homogenate were 96.5 and 95.7%, respectively.     

Colorimetric Analysis with N,N-Dimethyl-p-phenylenediamine of the Uptake of Intravenously Injected Horseradish Peroxidase by Various Tissues of the Rat

1. A method is described for the colorimetric determination of peroxidase with N,N-dimethyl-p-phenylenediamine. The amount of red pigment formed by peroxidase is proportional to the concentration of enzyme and to the time of incubation during the first 40 to 90 seconds. The influence of the concentration of enzyme, N,N-dimethyl-p-phenylenediamine, H₂O₂, the time of incubation, pH, the temperature, and the possible interference by oxidizing and reducing agents of tissues has been tested. 2. The method has been used to follow the uptake of intravenously injected horseradish peroxidase by 18 different tissues of the rat over a period of 30 hours. The highest concentration of the injected tracer enzyme was found in extracts of kidney, liver, bone marrow, thymus, and spleen. Considerable amounts were taken up by pancreas, prostate, epididymis, and small intestine. Lower concentrations were found in extracts of lung, stomach, heart, and skeletal muscle, aorta, skin, and connective tissue. No uptake was observed by brain and peripheral nerve tissue. 3. Tissue homogenates containing high concentrations of the injected peroxidase, in general also showed high or average activity of acid phosphatase. 4. Six hours after intravenous administration, the liver contained 27 per cent, the kidney 12 per cent, and the spleen, 1.4 per cent of the injected dose. 5. Approximately 20 per cent of the injected peroxidase was excreted in the urine during the first 6 hours, and the concentration of peroxidase in blood serum and urine fell exponentially during this time. After 6 hours, only low concentrations were excreted in the urine but low enzyme activity was still detectable after 30 hours. Approximately 6 per cent of the injected dose was excreted in the feces from 6 to 20 hours after administration. 6. After feeding through a stomach tube, low concentrations of peroxidase were found in blood serum and urine. Considerable variations in the extent of absorption from the gastrointestinal tract were observed in individual rats.     

Colloidal bismuth subcitrate and omeprazole inhibit alcohol dehydrogenase mediated acetaldehyde production by Helicobacter pylori

We have recently shown that Helicobacter pylori possesses marked alcohol dehydrogenase (ADH) activity and is capable - when incubated with an ethanol containing solution in vitro - of producing large amounts of acetaldehyde. In the present study we report that some drugs commonly used for the eradication of H. pylori and for the treatment of gastroduodenal diseases are potent ADH inhibitors and, consequently, effectively prevent bacterial oxidation of ethanol to acetaldehyde. Colloidal bismuth subcitrate (CBS), already at a concentration of 0.01 mM, inhibited H. pylori ADH by 93% at 0.5 M ethanol and decreased oxidation of 22 mM ethanol to acetaldehyde to 82% of control. At concentrations above 5 mM, CBS almost totally inhibited acetaldehyde formation. Omeprazole, a drug also known to suppress growth of H. pylori, also inhibited H. pylori ADH and suppressed bacterial acetaldehyde formation significantly to 69% of control at a drug concentration of 0.1 mM. By contrast, the H₂-receptor antagonists ranitidine and famotidine showed only modest effect on bacterial ADH and acetaldehyde production. We suggest that inhibition of bacterial ADH and a consequent suppression of acetaldehyde production from endogenous or exogenous ethanol may be a novel mechanism by which CBS and omeprazole exert their effect both on the growth of H. pylori as well as on H. pylori associated gastric injury.     

Purification of quinolinic acid phosphoribosyltransferase from rat liver and brain

Quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) was purified 3600-fold from rat liver and 280-fold from rat brain. Kinetic analyses (K_m = 12 μM for the substrate quinolinic acid and K_m 23 μM for the cosubstrate phosphoribosylpyrophosphate), physicochemical properties of the purified enzymes, inhibition by phthalic acid (K_i = 1.4 μM) and molecular weight determination (M_r 160 000 for the holoenzyme, consisting of five identical 32 kDa subunits) indicated the structural identity of quinolinic acid phosphoribosyltransferase from the two rat tissues. This was further confirmed immunologically, using antibodies raised against purified rat liver quinolinic acid phosphoribosyltransferase. Rat quinolinic acid phosphoribosyltransferase differs in several aspects from quinolinic acid phosphoribosyltransferase isolated from other organisms. The purified enzyme will prove a useful tool in the examination of a possible role of quinolinic acid in cellular function and/or dysfunction.     

A spectrophotometric procedure for determining the activity of various rat tissue oxidases.

A continuous spectrophotometric method suitable for the determination of the activities of several peroxisomal oxidases in rat tissue homogenates is described. The assay involves the continuous spectrophotometric measurement of the reaction product, H₂O₂, by coupling it to the reduction of a chromogen, o-dianisidine, with horseradish peroxidase. Catalase interference was overcome using azide to inhibit its activity and a H₂O₂ standard curve used to quantitate oxidase activity in terms of microkatals per milliliter of enzyme.