NAD Oxidoreductase Research Articles

Über den Abbau von L-Äpfelsäure durch Hefen verschiedener Gattungen mit Malatenzym

(1) The aerobic assimilation of malic acid is not a character of certain yeast genera or species as was shown by testing more than 300 different strains. Single strains of the following-species were found to grow on malic acid as the only carbon source: Candida pulcherrima, C. utilis, C. mycoderma, Torulopsis famata, Pichia membranaefaciens, P. wickerhamii, Hansenula capsulata, Trigonopsis variabilis , and Zygosaccharomyces chevalieri . (2) During fermentation C. pulcherrima and T. famata decompose up to 40% and C. utilis up to 80% of the L-malic acid that is present in the medium. (3) L-Malic acid is decomposed to CO 2 and the corresponding amounts of ethanol or pyruvate by cell free extracts of yeasts of the genera Pichia, IIansenula, Trigonopsis, Zygosaccharomyces, Kloeckera, Candida , and Torulopsis . (4) The enzymatic activity of malate dehydrogenase (L-malate: NAD oxidoreductase, E.C.1.1.1.37) was separated from the decarboxylating enzyme by gel chromatography of extracts of some strains of the genera Pichia, Candida , and Hansenula . It is assumed that malic acid is decomposed by a malic enzyme (L-malate: NAD oxidoreductase, decarboxylating E.C. 1.1.1.38) that was partially purified and found to have a molecular weight of 120000–130000. (1) Die Untersuchung der aeroben Verwertung von L-Äpfelsäure bei über 300 Hefestämmen verschiedener Gattungen und Arten ergab, daß diese Eigenschaft nicht gattungs- oder artipezifisch ist. Einzelne Stämme folgender Arten wuchsen aerob mit Äpfelsäure als einziger Kohlenstoffquelle: Candida pulcherrima, C. utilis, C. mycoderma, Torulopsis famata, Pichia membranaefaciens, P. wickerhamii, Hansenula capsulata, Trigonopsis variabilis und Zygosaccharomyces chevalieri . (2) Unter Gärbedingungen wurden unterschiedliche Mengen von L-Äpfelsäure abgebaut. C. pulcherrima und T. famata vermögen bis zu 40% und C. utilis bis zu 80% der im Medium vorhandenen L-Äpfelsäure umzusetzen. (3) Zellfreie Extrakte aus Hefen der Gattungen Pichia, Hansenula, Trigonopsis, Zygosaccharomyces, Kloeckera, Candida und Torulopsis bilden aus L-Äpfelsäure in Abhängigkeit von Mn ++ und NAD CO 2 und in entsprechenden Mengen Äthanol oder Pyruvat. (4) Bei einigen Stämmen der Gattungen Pichia, Candida und Hansenula konnte durch Gel-Chromatographie die Äpfelsäure decarboxylierende Aktivität von der Malat-Dehydrogenase (L-Malat: NAD Oxidorekdutase E.G.1.1.1.37) abgetrennt und angereichert werden, woraus auf das Vorkommen eines Malatenzyms (L-Malat: NAD Oxidoreduktase, decarboxylierend E.C.1.1.1.38) geschlossen werden kann. Das Molekulargewicht dieses Enzyms liegt bei 120-130000.

Alcohol dehydrogenase from Thea sinensis seeds.

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E. C. 1. 1. 1. 1.) from Thea sinensis seeds (variety: Zairai) was isolated and purified about 1, 500-fold using preparative disc electrophoresis. The specific activity was about 3.4units/mg protein against ethyl alcohol. Its s020. w value was 6.96S and its molecular weight was approximately 150, 000 using gel filtration on Sephadex G-200. The physical, chemical and catalytic properties of the enzyme are described. The oxidoreduction products formed by the enzyme were identified by gas chromatography, and for the unsaturated compounds the conversion of double bond and geometrical isomerization was observed. The substrate specificity of tea enzyme is discussed in comparison with the enzymes from Leuconostoc mesenteroides, and horse and human livers. Paticularly a tendency for reactivity in the oxidoreduction of unsaturated alcohols and aldehydes were discribed by comparing the effects of geometry, the position of the double bond and the length of chain in substrates.

Pitfalls in histochemical localization studies of NADPH generating enzymes or enzyme systems in rat small intestine.

In the presence of excess β-glycerophosphate or p-nitrophenylphosphate NADPH diaphorase of the epithelial cell in rat small intestine has its highest activity in the villi at the basis of the microvilli, where smooth endoplasmic reticulum of the cells is present. In the absence of β-glycerophosphate or p-nitrophenylphosphate the diaphorase activity is much higher and broader localized in crypts and villi. The increased activity is due to the conversion of NADPH to NADH by non-specific (mostly alkaline-) phosphatase activity, so that both NADPH and NADH diaphorase activities are measured. When NADPH is generated by a specific NADP+-linked dehydrogenase, such as glucose-6-phosphate dehydrogenase, and nitroblue tetrazolium is present to trap the reducing equivalents formed, the histochemical localization of the dehydrogenase is identical to that of NADPH diaphorase, although the dehydrogenase may be present in another cell compartment (glucose-6-phosphate dehydrogenase for instance has a cytosolic distribution). Therefore the localization of a dehydrogenase may be falsely interpreted histochemically, according to the diaphorase reaction involved. A different localization may be obtained when the diaphorase reaction is circumvented by the addition of an alternative hydrogen carrier, such as phenazine methosulphate. Also in coupled dehydrogenase assays this may be observed. The hexokinase reaction, coupled to the glucose-6-phosphate dehydrogenase reaction in the presence of nitroblue tetrazolium and the absence of phenazine methosulphate, has a distribution identical to NADPH diaphorase. In the presence of phenazine methosulphate the enzyme has an almost ubiquitous distribution in the small intestinal epithelial cell. When a substrate may react both with NADP+- and NAD+-linked dehydrogenases, such as L-malate: NADP oxido-reductase (decarboxylating) and L-malate: NAD oxidoreductase respectively, the high activity of intestinal alkaline phosphatase may influence the histochemical distribution by converting part of NADP+ to NAD+. The addition of another phosphate ester, such a β-glycerophosphate or p-nitrophenylphosphate, may therefore influence the observed distribution.

Some observations on serum lactate dehydrogenase (pyruvate→ lactate) assays optimised at 37°

One hundred and four sera were assayed for serum lactate dehydrogenase ( l-lactate : NAD oxidoreductase, EC 1.1.1.27.) activity (pyruvate→ lactate) by three methods: the Henry assay used at 37°, and the McQueen and Eskalab assays which are claimed to be optimised at 37°. The McQueen assay gave significantly less lactate dehydrogenase activity, due to substrate inhibition, than the other two methods which appeared to be equivalent. An excess proportion of M-type lactate dehydrogenase subunits in samples was slightly overestimated by both the Eskalab and McQueen assays. It was concluded that an assay using pyruvate concentrations between 0.6 mM (Henry assay) and 1.5 Mm (Eskalab assay) would be more satisfactory.

3-Hydroxy steroid dehydrogenase activities of cortisone reductase.

The behaviour of various C(19) and C(18) steroids as substrates for crystalline preparations of cortisone reductase (EC 1.1.1.53) is described. 3alpha(Axial,3R)-, 3alpha(equatorial,3R)- and 3beta(axial,3S)-hydroxy steroid-NAD oxidoreductase activities are demonstrated. Four pairs of the substrates differed only in the shape of the a/b ring junction, three pairs differed only in substitution at C-10, and four pairs differed only in substitution in ring d. The shape of the substrate molecule and certain substituents (e.g. 10beta-methyl, 17beta-hydroxy, 16-oxo or 17-oxo) altered substrate behaviour, but steroids differing considerably in shape nevertheless acted as substrates, suggesting the possibility of a large or flexible binding site. K(m) values varied about 10-fold, many being approx. 140mum. V(max.) values covered a greater range (about 200-fold) and the good substrates had high V(max.) values rather than low K(m) values.

Purification and characterization of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from a nitrogen-fixing bacterium.

Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP(+) reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP(+) reductase in the photochemical reduction of NADP(+) by blue-green algal particles. The ferredoxin-NADP(+) reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP(+) was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD(+) transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (K(m) = 5.0 x 10(-3)M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase.

Modification of pig heart lactate dehydrogenase: A derivative possessing enhanced thermal stability

Pig heart lactate dehydrogenase ( l-lactate:NAD oxidoreductase, EC 1.1.1.27) has been modified by reaction with pentanedial to give a water-soluble product. This derivative retains 50% of initial enzymic activity, and its K m for the reduction of pyruvate is close to that of the native enzyme. Reaction with pentanedial markedly increases the thermal stability of the enzyme at pH 5.0–6.0 and at pH 9.0.

Localization of 3-β-hydroxysteroid dehydrogenase in the inner membrane subfraction of rat testis mitochondria

1. 1. The activity of 3-β-hydroxysteroid dehydrogenase (3-β-hydroxysteroid: NAD (P) oxidoreductase, EC 1.1.1.51) in rat testis mitochondria and submitochondrial fractions was determined. 2. 2. Innr and outer mitochondrial membranes were obtained by subjecting the mitochondrial pellets to controlled osmostic lysis, ultrasonic vibration, and treatment with a non-ionic detergent, Lubrol WX, followed by differential centrifugation. 3. 3. The purity of the isolated mitochondria and of mitochondrial membranes was ascertained by electron microscopy, measurement of enzyme markers and by a method based upon labelling of the mitochondrial and cytoplasmic RNA with [5 −3H]orotic acid. 4. 4. The results of this study suggest that the 3-β-hydroxysteroid dehydrogenase is present in rat testis mitochondria, free of endoplasmic reticulum and that this enzyme is mainly localized in the inner mitochondrial membrane.

EFFECTS OF UREA ON THE ACTIVITY OF GLUTAMATE DEHYDROGENASE, GLUTAMINE SYNTHETASE, CARBAMYL PHOSPHATE SYNTHETASE, AND CARBAMYL PHOSPHOKINASE IN RUMINANT TISSUES

The effect of an isonitrogenous (2.7% N) semipurified diet containing 0, 50, or 100% of the added nitrogen (N) as urea (comprising 0, 39, or 76% of the total N in the diet) on the activities of major enzymes involved in nitrogen metabolism was investigated with calves. The activities of glutamate dehydrogenase (L-glutamate:NAD(P) oxidoreductase (deaminating) enzyme code (EC) 1.4.1.3), glutamine synthetase (L-glutamate:ammonia ligase (ADP) EC 6.3.1.2), carbamyl phosphate synthetase (ATP:carbamate phosphotransferase (dephosphorylating) EC 2.7.2.5), and carbamyl phosphokinase (ATPcarbamate phosphotransferase EC 2.7.2.2) were assayed in the liver, rumen epithelial tissue, and rumen microorganisms. No significant differences among diets in the activity of glutamate dehydrogenase from liver or rumen epithelium were found. Rumen fluid (rumen microorganisms) samples from calves fed the 50% urea-N diet exhibited higher glutamate dehydrogenase activity than the 0% urea-N or 100% urea-N diets. Rumen epithelium glutamine synthetase activities were slightly higher for the 50% urea-N diet than the 0 or 100% urea-N diets. Liver glutamine synthetase activities were also slightly higher for the urea-containing diets when compared with the 0% urea-N diet. Rumen microorganisms glutamine synthetase activity was significantly (P < 0.05) higher for animals fed the 50% urea-N diet than those fed 0 or 100% urea-N diets. Liver carbamyl phosphate synthetase was significantly (P < 0.01) higher than rumen epithelium or phosphokinase of rumen microorganisms but there were no significant differences among diets. Rumen fluid from calves fed the 50 or 100% urea-N diets had higher carbamyl phosphokinase activity than animals fed the 0% urea-N diet.

Microbial assimilation of methanol. The ethanol- and methanol-oxidizing enzymes of the yeast Candida boidinii.

An NAD‐dependent alcohol dehydrogenase has been isolated from the yeast Candida boidinii grown on methanol. A study of the properties of the enzyme has shown that it is very similar to the alcohol: NAD oxidoreductase from baker's yeast. The alcohol dehydrogenase from Candida boidinii does not catalyze the oxidation of methanol and is constitutively formed. A mutant, 48, has been isolated which is unable to grow on methanol as sole carbon and energy source. This strain lacks the methanol‐oxidizing enzyme but not NAD‐dependent alcohol dehydrogenase. Cells grown on glucose or ethanol do not contain the methanol‐oxidizing enzyme. During adaptation from glucose to methanol, enzyme activity appears before growth on methanol begins. The methanol‐oxidizing enzyme has been partially purified. It catalyzes the oxidation of methanol to formaldehyde and hydrogen peroxide, and is independent of the addition of a hydrogen acceptor. The prosthetic group of this enzyme is FAD. The molecular weight was calculated to be 600000, one subunit has a molecular weight of 74000.The optima of pH and temperature for enzyme activity are 7.5–9.5 and 30°C, respectively. It is unstable in acidic pH.The enzyme is not specific for methanol. It also oxidizes lower primary alcohols. The Km value for methanol is 2.0 mM and that for ethanol 4.5 mM.

Oxidation of acetaldehyde by rat-liver mitochondria in relation to ethanol oxidation and the transport of reducing equivalents across the mitochondrial membrane.

The primary product of the well‐known oxidation of acetaldehyde by rat liver mitochondria is acetate. About 50% of the acetate formed from acetaldehyde is converted to ketone bodies under the experimental conditions used. The oxidation of acetaldehyde by rat liver mitochondria is catalyzed mainly by an aldehyde: NAD oxidoreductase present in the matrix compartment of the mitochondria. About 20% of the total mitochondrial acetaldehyde dehydrogenase activity may be confined to the outer membrane or the intermembrane space. The enzyme has a broad substrate specificity, pH‐optimum at about pH 9.0 and is inhibited by tetraethylthiuram disulfide, chloral hydrate and arsenite. The enzyme is steroid‐insensitive.Two Km‐values for acetaldehyde, below 0.1 μM and 1.0 mM, and two Km‐values for NADM+, 2 μM and 50 μM, were obtained with a partially purified 100000 ×g supernatant. The presence of two aldehyde dehydrogenases in rat liver mitochondria could not be shown. Oxidation of acetaldehyde by rat liver mitochondria causes an inhibition of the production of 14Co2, from [14C]palmitate. The inhibition can be accounted for by dilution of the acetyl‐coenzyme A pool of the mitochondria.The 3‐hydroxybutyrate/acetoacetate ratio during acetaldehyde oxidation is kept at the same level as during oxidation of oleate plus coenzyme A. The mitochondrial acetaldehyde dehydrogenase activity is 80% of the total acetaldehyde dehydrogenase activity of rat liver. The results are discussed with regard to the oxidation of acetaldehyde and to the transfer of reducing equivalents from cytosol to mitochondria during ethanol metabolism.

Genetics of Glycerol-3-phosphate Dehydrogenase Isozymes in White Muscle of Lake Whitefish (Coregonus clupeaformis)

Multiple isozymes of glycerol-3-phosphate dehydrogenase (L-gIycerol-3-phosphate: NAD oxidoreductase, E. C. 1.1.1.8) have been resolved by starch gel electrophoresis of extracts of muscle tissue from lake whitefish (Coregonus clupeaformis). The isozyme electropherograms show that three kinds of subunit, A, B, and C, are synthesized in red muscle and two of these, A and B, are also found in white muscle. In red muscle the subunits evidently combine to form catalytically active dimers of the following types: AA, AB, BB, BC, and CC. In white muscle only the AA, AB, and BB dimers were observed.A genetic and molecular structure model is proposed for the glycerol-3-phosphate dehydrogenase (G-3-PDH) isozymes in white muscle of lake whitefish. On the basis of two alleles for A subunits and three alleles for B subunits, the model predicts a total of 18 distinct, electrophoretic G-3-PDH phenotypes for all possible AA, AB, and BB dimeric isozymes. The model has been confirmed by the results of a breeding experiment that tested the heritability of each of the five known alleles. A difference in the rate of heat inactivation of AA and BB isozymes was also interpreted as additional evidence for the unique genetic and molecular nature of the two kinds of subunits.Surveys of natural populations of lake whitefish revealed some marked variations in the frequencies of G-3-PDH alleles in fish from different geographical areas.The cisco (Coregonus artedii) also appears to have three "b" alleles for G-3-PDH.

Evidence for the existence of lactate dehydrogenase N in embryonic chick liver

Lactate dehydrogenase ( l-lactate:NAD oxidoreductase, EC 1.1.1.27) from embryonic chick liver had different kinetic and thermal stability properties from lactate dehydrogenase from adult chick liver, and lactate dehydrogenase prepared from different sources known to contain varying percentages of the lactate dehydrogenases 1, 2, 3, 4 and 5 isozymes. The embryonic chick liver lactate dehydrogenase could not be separated from adult chick liver lactate dehydrogenases or lactate dehydrogenase 5 on the basis of its electrophoretic mobility on polyacrylamide disc gels. However, embryonic chick liver lactate dehydrogenase utilized α-ketoglutaric acid as a substrate, and 3-acetylpyridine NAD as a coenzyme more efficiently than any of the other lactate dehydrogenase isozymes. Also embryonic chick liver lactate dehydrogenase was more resistant to heating at 65°C than the other lactate dehydrogenase isozymes. Further experiments showed that the kinetic properties of embryonic chick liver lactate dehydrogenase were consistent with those demonstrated for lactate dehydrogenase X.

Partial purification and properties of UDPG dehydrogenase from Escherichia coli

1. 1. UDPG dehydrogenase (UDPG: NAD oxidoreductase, EC 1.1.1.22), was purified 88-fold to a specific activity of 2.3 from a derepressed strain of Escherichia coli. 2. 2. The enzyme has a molecular weight of approximately 86 000 and a pH optimum of 9.0; K m is 1.0 mM for UDPG and 0.05 mM for NAD, respectively. 3. 3. UDPG, CDPGlc and dTDPGlc, but not GDPGlc or ADPGlc function as substrates. 3-Acetylpyridine adenine dinucleotide, 3-pyridinealdehydeadenine dinucleotide, thionicotinamide adenine dinucleotide, deamino adenine dinucleotide and 3-acetylpyridine deamino adenine dinucleotide, but not 3-pyridinealdehyde deaminoadenosine dinucleotide, are cosubstrates. 4. 4. The enzyme is stable for 2 months when frozen at −4 °C in the presence of UDPG. UDPG and 2-mercaptoethanol are necessary for optimal enzyme activity. 5. 5. UDPXyl is an inhibitor strictly competitive with UDPG and noncompetitive with NAD; NADH is an inhibitor strictly competitive with NAD and uncompetitive with UDPG. UDPglucuronate is an inhibitor competitive with UDPG at high UDPG concentrations; at low UDPG concentrations UDPglucuronate is an inhibitor which displays positive cooperativity.

Pentitol oxidoreductases in Sclerotinia sclerotiorum.

Pentitol: NADP and pentitol: NAD oxidoreductase were detected in cell-free extracts of mycelia and sclerotia ofSclerotinia sclerotiorum (Lib.) D By. grown on synthetic liquid medium containingd-xtlose,l-arabinose ord-ribose. Both enzymes were similar to those reported from other fungi. The enzymes were not detected when the fungus was grown on a medium containingd-glucose.

The bond hydrolyzed by cardiolipin-specific phospholipase D

Cardiolipin-specific phospholipase D from Hemophilus parainfluenza hydrolyzes cardiolipin to yield phosphatidic acid and phosphatidyl-1'- sn-glycerol. In order to determine the configuration of the latter compound it was hydrolyzed by phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). The water-soluble product was identified as X-glycerol 1-phosphate by chromatography in two systems and oxidation with periodate to yield formaldehyde and glycolaldehyde phosphate. The failure to react with glycerol phosphate dehydrogenase ( l-glycerol-3-phosphate: NAD oxidoreductase, EC 1.1.1.8) specified that the compound must be sn-glycerol 1-phosphate. The results demonstrate that cardiolipin-specific phospholipase D has an absolute specificity for the bond between phosphate and the C-3' of cardiolipin's central glycerol.

Horse liver 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase as a liver aldehyde dehydrogenase

3α,7α,12α-Trihydroxy-5β-cholestan-26-al dehydrogenase (3 α,7 α,12 α-trihydroxy-5β-cholestan-26-al + NAD + + H 2O → 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid + NADH + H +) in a soluble fraction of horse liver was studied. 1. 1. Horse liver 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase consists of two components, the major one of which was purified about 70-fold by (NH 4) 2SO 4 fractionation, gel filtration, ion-exchange column chromatography and hydroxylapatite column chromatography. 2. 2. 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase was always accompanied by liver aldehyde:NAD oxidoreductase (EC 1.2.1.3) activity, and the ratio of these two enzyme activities was not altered by purification. 3. 3. Both 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase and horse liver aldehyde:NAD + oxidoreductase were reduced to a similar extent on inactivation of the purified enzyme by heating or by incubation with p-chloromercuribenzoate. 4. 4. The K m value for NAD + of 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase was found to be roughly equal to that of liver aldehyde:NAD + oxidoreductase. 5. 5. The possibility was discussed that both 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase and liver aldehyde:NAD + oxidoreductase activities may be due to a single protein.

Studies on the mechanism of action of UDP- D-glucose dehydrogenase from beef liver. II

Uridine 5′-(α- D-glucopyranosyl pyrophosphate) (UDP- D-glucose) was converted into UDP- D-glucuronic acid by pure bovine UDP- D-glucose:NAD oxidoreductase (E.C. 1.1.1.22) in H 2 18O. The D-glucosyluronate moiety of the UDP- D-glucuronate formed in the reaction was shown by mass spectrometry to contain 18O. UDP- D-glucuronate carboxy-lyase (E.C. 4.1.1.35) was then used to convert the UDP- D-glucuronic acid ( 18O) into UDP- D-xylose; the D-xylosyl moiety of the latter contained no 18O when examined by mass spectrometry. These data indicate that, in the NAD-linked conversion of UDP- D-glucose into UDP- D-glucuronic acid, the new oxygen atom is derived from water and is located at C-6 of the D-glucosyluronate moiety of UDP- D-glucuronic acid. Furthermore, a mass-spectral fragmentation scheme is presented for per- O-trimethylsilyl-β- D-glucuronic acid, based on high-resolution, mass-spectral measurements.

Effects of 4-hydroxybutyric acid on the mitochondrial respiratory system. The mechanism of the inhibition of glutamate oxidation

1. 1) Gamma-hydroxybutyric acid (4-OHB) inhibit O 2 uptake by rat liver mitochondria respiring with glutamate, without a change in P/o ratio. This compound does not modify O 2 uptake in mitochondria respiring with 2-oxoglutarate and succinate while it inhibits O 2 uptake stimulated by fum a rate and malate, without a change in P/O ratio. 2. 2) In liver mitochondria respiring with glutamate in the presence of 4-OHB the inhibition of aspartate formation can account for that of O 2 uptake. Both NH 3 production and O 2 uptake in the presence of malonate are unaffected. Fumarate, malate, or oxaloacetate are each able to neutralize the inhibitory effect of 4-OHB on O 2 uptake and on aspartate formation in rat liver mitochondria incubated with glutamate. 3. 3) Mitochondria purified from kidney, brain and heart are able to oxidize glutamate but exhibit a differential sensitivity to the action of 4-OHB; brain and kidney mitochondria oxidizing glutamate are inhibited by 4-OHB to a lesser degree than liver mitochondria. In heart mitochondria the oxidation of glutamate is not affected by 4-OHB. 4. 4) In submitochondrial liver particles 4-OHB did not inhibit either the transamination between glutamate and oxaloacetate, or the oxidation of malate to oxaloacetate; negative findings were also obtained by employing purified enzymes L-aspartate-2-oxoglutarate amino transferase (EC 2.6.1.1.) or L-malate: NAD oxidoreductase (EC 1.1.1.37) .

Effects of elevated glucose concentrations on the metabolism of the aortic wall.

The effects of elevated glucose concentrations on the metabolism of the aortic wall were examined in a preparation of tubular segments of rabbit descending thoracic aorta comprised of intima and media only. Increased medium glucose concentrations (20-50 mm) resulted in increased aortic sorbitol and fructose concentrations and an increased rate of fructose release into the medium. This increased flux through the polyol pathway can be explained as a consequence both of an increased free intracellular glucose concentration and of the kinetic characteristics of the alditol: NADP oxidoreductase and the l-iditol: NAD oxidoreductase isolated and partially purified from rabbit thoracic aorta. Incubation with elevated glucose concentrations for 2 or more hr was also associated with a significant increase in the water content of the tissue without a significant increase in the inulin space. The oxygen uptake of the tissues incubated with elevated glucose concentrations was significantly reduced; this appears to result from a limitation imposed by oxygen diffusion at physiological oxygen tensions. A compensatory increase in glycolysis and an increase in the aortic lactate/pyruvate concentration ratio were also observed. The oxygen uptake and lactate production of tissue incubated with 50 mm glucose could be preserved at rates observed in tissue incubated with a physiological glucose concentration by the addition of 40 mm mannitol to the medium. Aortic intima and media from alloxan-diabetic rabbits also exhibit an increased water content and a decreased rate of oxygen uptake. These observations suggest that elevated ambient glucose concentrations result in significant alterations in the metabolism of aortic intima and media.