Further purification and characterization of diacetyl reducing enzymes from beef liver

• 1.1. Two diacetyl reducing enzymes have been isolated from beef liver. One of them, a monomer of mol. wt 28-30,000 dalton and pI 6.2, corresponds to the low moleclular weight diacetyl reductase formerly accounted for using preparations of this organ; it has been now identified as an l-glycol dehydrogenase.
• 2.2. The other one, an oligomer of 78,000 dalton and pI 7.0, which matches the high molecular weight diacetyl reductase, is, in the authors' opinion, a new enzyme for which the systematic name L(+)-α-hydroxycarbonyl: NAD(P) oxidoreductase (EC 1.1.1...) and common name α-dicarbonyl reductase are proposed.

     

Mitochondrial malic enzyme from the crayfish abdomen muscle,purification, regulatory properties and possible physiological role

• 1.1. Mitochondrial malic enzyme (l-Malate: NADP oxidoreductase (oxaloacetate decarboxylating) EC 1.1.1.40) has been isolated from abdomen muscle of crayfish Orconectes limosus by chromatography on Sepharose 6B and DEAE cellulose. Specific activity of the purified enzyme was about 5 μmols per min per mg protein, which corresponds to about 30-fold purification.
• 2.2. This enzyme showed extremely small reversiblity, since the reaction in the direction of decarboxylation is at least 37, 190 and 760 times that for the carboxylation at pH 7.0, 7.5 and 8.0 respectively.
• 3.3. Purified enzyme showed allosteric properties, which was more accentuated at more alkaline pH (Hill coefficients were 1.1, 1.7 and 1.8 at pH 7.0, 7.5 and 8.0 respectively). The activity of malic enzyme was increased considerably in the presence of succinate and fumarate.
• 4.4. Mitochondira isolated from abdomen muscle of Orconectes limosus incubated in the presence of malate, fumate and succinate catalysed pyruvate production which was stimulated by ADP and inhibited by respiratory chain inhibitors.
• 5.5. NADH but not NADPH oxidation was catalysed by broken mitochondria or sonic particles. When NADPH and NAD were added simultaneously the rate of oxidation. This suggests the presence of active NADPH:NAD transhydrogenase in mitochondria isolated from the crayfish abdomen muscle.
• 6.6. A possible metabolic role for NADP-linked malic enzyme/transhydrogenase couple in abdomen muscle of crayfish Orconectes limosus is proposed.

     

Reductive metabolism of D-glyceraldehyde,L-glyceraldehyde and dihydroxyacetone in rat liver: Identification of alcohol dehydrogenase as the main responsible enzyme

• 1.1. The paper describes NADH- and NADPH-dependent enzyme activities in rat liver which catalyse the reduction of the following substrates: d-glyceraldehyde, l-glyceraldehyde and dihydroxyacetone. Test conditions for the optimal rates of the oxidoreductase reactions are described.
• 2.2. As a test of metabolic relevance of these activities the hormonal status of the rats was changed by pretreatment with alloxan.
• 3.3. This lowers all described activities if the concentration of blood glucose is increased. But there is also a range of elevated activities which are not associated with changes in the glucose concentration.
• 4.4. It is shown that rat liver alcohol dehydrogenase (EC 1.1.1.1) 2 is the enzyme which catalyses the reduction of the substrates named above and also of acetaldehyde with NADH and NADPH. The preparation and characterization of the enzyme are described.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

• 1.1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme.
• 2.2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium.
• 3.3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate.
• 4.4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M.
• 5.5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme.
• 6.6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Inhibition of dihydrofolate reductase by palmitoyl coenzyme A

• 1.1. Palmitoyl-CoA was found to inhibit bovine liver dihydrofolate reductase.
• 2.2. 50% inhibition of the enzyme was obtained with 1.5 μM palmitoyl-CoA.
• 3.3. The inhibition was reversed by addition of bovine serum albumin, β-cyclodextrin or spermine.

     

Interaction of steroids with d-amino acid oxidase

• 1.1. Progesterone hibited d-amino acid oxidase (d-amino acid : O₂ oxidoreductase (deaminating), EC 1.4.3.3.) in competition with its substrate, d-alanine, Binding of progesterone brought about the increase in both fluorescence intensity and fluorescence polarization of FAD, which indicates that the environment surrounding FAD chromophore is modified due to a conformational change in the apoenzyme.
• 2.2. Ethinyl estradiol testosterone, testosterone propionate, corticosterone and aldosterone also inhibited the enzyme slightly in the same manner. Their binding also produced a slight increase in FAD fluorescence without decreasing the fluorescence polarization.
• 3.3. Cholesterol did not inhibit the enzyme, though it increased the fluorescence polarization of FAD. This indicates the binding of cholesterol with the enzyme at a site other than the substrate binding site.

     

Some properties of the NADP-malic enzyme from mango fruit,Mangifera indica

• 1.1. Malic enzyme l-malate-NADP-oxidoreductase (oxaloacetate-decarboxylating) (EC 1.1.1.40) was located in the cytosolic fraction of ripening mango fruit.
• 2.2. The purified enzyme has an isoelectric point of 6.86 and an activation energy of 11.9kcal/mol.
• 3.3. The amino acid composition of the enzyme was determined and revealed a low cysteine and tryptophan content.
• 4.4. The enzyme has an ultraviolet absorption maximum at 266 nm with maxima for fluorescence excitation and emission at 285 and 328 nm.
• 5.5. The enzyme shows positive cooperativity between the malate binding sites and the effect of allosteric regulators and structural analogues on the activity were investigated.

     

Electron transport in sheep (Ovis aries) liver microsomes. Characteristics,effect of lipid peroxidation and aqueous acetone extraction

• 1.1. Activities and contents of the electron transport components of sheep (Ovis aries) liver microsomes are given. Enzymes or enzyme systems assayed are NADH-cytochrome c reductase, NADH-ferrieyanide reductase, NADH-dichlorophenol-indophenol reductase, NADH- and NADPH-neotetrazolium reductase, cytochrome b₅, cytochrome P-450 and the cyanide binding protein.
• 2.2. Prior lipid peroxidation of sheep liver microsomes did not markedly alter NADH- and NADPH-cytochrome c reductase or NADH-ferricyanide reductase activities but decreased NADPH-dependent aniline hydroxylation activity. Intermediate amounts of prior lipid peroxidation enhanced the activity of NADPH-dependent lipid peroxidation.
• 3.3. NADH-cytochrome c reductase activity of sheep liver microsomes was decreased 39–56% when 60% of the microsomal organic phosphorus was removed by acetone:water 90:10 (v/v) extraction but was not markedly altered by the removal of 25 and 44% of the microsomal organic phosphorus by acetone:water 100:4 (v/v) and acetone:water 100:7 (v/v) extractions.

     

Studies on glycogenolysis in carp liver: Evidence for an amylase pathway for glycogen breakdown

• 1.1. Glycogen-phosphorylase seems to be lacking in the carp liver. This enzymatic defect bears a resemblance to glycogen storage disease type VI, described in humans.
• 2.2. Carp liver homogenates exhibit an important γ-amylase (α-glucosidase, EC 3213) activity. By its pH curve and distribution in subcellular fractions of liver, this enzyme could be, to a large extent, of lysosomal origin.
• 3.3. During the strong hepatic glycogenolysis, which is induced in carp by insulin injections, the γ-amylase pathway could offer an explanation for glycogen breakdown in a tissue where glycogen phosphorylase is supposed to be absent.

     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

Purification,characterization and kinetic mechanism of glucose-6-phosphate dehydrogenase from mouse liver

• 1.1. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) from mouse liver has been purified 1100-fold by extraction, ion-exchange chromatography on DE-52, absorption chromatography on Bio-Gel HTP and gel filtration through sepharose 6 HR 10/30. The purified enzyme showed a single band in silver stained SDS-PAGE.
• 2.2. The native and subunit molecular weight were 117 and 31 kDa respectively.
• 3.3. The kinetic studies and the patterns obtained from the inhibition by-products suggest that the enzyme follows an ordered sequential kinetic mechanism.
• 4.4. The reduced K_m values for the substrates favour the operativity of the enzyme. The “fine control” of the enzymatic activity was exerted by the NADPH, whose K_i is several fold lower than the in vivo concentration.

     

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

• 1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.
• 2.2. The maximum pH of the reaction in the liver microsomes was 7.6.
• 3.3. The K_m and V_max values for all-trans, 9-cis and 13-cis-retinals were determined.
• 4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.
• 5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.
• 6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b₅ IgG.
• 7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.
• 8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.
• 9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.
• 10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.

     

Fatty acid synthetase from pigeon red blood cells

• 1.1. Fatty acid synthetase has been purified 200-fold from pigeon erythrocytes.
• 2.2. The enzyme gave 2 major staining bands on disc gel electrophoresis corresponding to the complex and dissociated forms of the enzyme.
• 3.3. Sucrose density gradient centrifugation of the enzyme showed only one sedimenting peak and high performance liquid chromatography also showed only 1 major light absorbing peak.
• 4.4. The molecular weight of the enzyme was estimated to be 300,000–330,000 and the enzyme is comprised of 2 subunits of similar molecular weights.
• 5.5. The red blood cell fatty acid synthetase was found to be immunochemically nonidentical with the liver fatty acid synthetase.

     

A fluorescence study of substrate and inhibitor binding to bovine liver dihydrofolate reductase

• 1.1. Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with K_i = 11 nM.
• 2.2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme.
• 3.3. The dissociation constants (K_d) of MTX, FMTX, NADPH and 7,8-dihydrofolate (DHF) from bovine liver DHFR have been determined by fluorometric titrations.
• 4.4. The K_d values for NADPH, MTX and FMTX from the complementary binary complexes (MTX·DHFR, FMTX·DHFR and NADPH·DHFR) were also obtained; these show a 2- to 4-fold decrease with respect to those obtained by titration of the free enzyme.
• 5.5. A competitive assay for MTX has been developed by exploiting the fluorescence enhancement of DHFR-bound FMTX. This assay may be useful for the routine determination of MTX in the concentration range from 10⁻⁹ to 10⁻⁷ M.

     

Effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail

• 1.1. The effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail were investigated.
• 2.2. The level of liver NAD was not affected by niacin deficiency whereas the level of pectoral muscle NAD was markedly reduced.
• 3.3. In all dietary treatments the liver had the highest turnover rates of proteins, heart and brain had intermediate rates, and pectoral muscle had the lowest rates.
• 4.4. Relative turnover rates of proteins in all tissues (particularly pectoral muscle) of the niacin deficient group were significantly higher than those of pair-fed control group, although there were no significant differences in turnover rate between pair-fed control and control groups.
• 5.5. The high turnover rate of proteins in niacin deficiency was primarily attributed to enhanced degradation rate of proteins rather than enhanced synthesis rate of proteins.
• 6.6. Optical density scanning (or densitometric) of water-soluble pectoral muscle proteins separated by isoelectric focusing revealed several additional minor protein bands between major protein bands in the niacin deficient group which were more pronounced in the acidic region of the gel.
• 7.7. These results suggest that proteins with a low pI value in pectoral muscle of the niacin deficient animal are highly sensitive to protein degradation.

     

An alkaline phosphatase from Thermus sp strain Rt41A

• 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
• 2.2. The enzyme has a M_r of 160,000 and is trimeric.
• 3.3. The half-life of the enzyme is 5 min at 85°C.
• 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
• 5.5. The H_m of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
• 6.6. The enzyme is inhibited 50% by 20 mM Ca²⁺ or Mg²⁺.
• 7.7. The K_i for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
• 8.8. Urea (200 mM) is not inhibitory.

     

Regulation of horse-liver glutathione reductase

• 1.1. The enzyme was rapidly inactivated by NAD(P)H, GSH, dithionite or borohydride, while activity increased in the presence of NAD(P)+ or GSSG. NADH was more efficient for inactivation than NADPH. Redox inactivation required neutral or alkaline pH, was maximal at pH 8.5, and depended on the presence of metal cations.
• 2.2. GSSG and dithiothreitol fully protected the enzyme from inactivation at concentrations stoichiometric with NAD(P)H. Ten-fold higher ferricyanide or GSH concentrations were required to obtain partial protection. NAD+ or NADP+ were quite ineffective.
• 3.3. GSSG fully reactivated the inactive enzyme at 38°C and neutral to acidic pH (5.5–7.5). Reactivation by dithiothreitol was accomplished in short periods of time at pH 8.5 although the activity was progressively lost afterwards. Ferricyanide and GSH also reactivated the enzyme to different extents.

     

Purification and properties of aldehyde dehydrogenase from Aspergillus nidulans

• 1.1. Aspergillus nidulans produces aldehyde dehydrogenase (ALD-DH) only when grown in the presence of ethanol, threonine or acetoacetic acid as inducer. Enzyme formation is inhibited by glucose in the growth medium.
• 2.2. ALD-DH is purified by a rapid procedure using Cibacron Blue Affinity Chromatography with specific inhibitoe elution by NAD plus 2:2′ dithiodipyridine or 2:4 disulfiram.
• 3.3. The pure native enzyme has a M_r=265,000 and a subunit M_r of 540,000. Its optimum pH is 8.5; its preferred substrate is acetaldehyde and it can use either NAD or NADP.