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.

     

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.

     

Involvement of lysine residue in the nucleotide binding of pigeon liver malic enzyme: modification with affinity label periodate-oxidized nadp

• 1.1. Periodate-oxidized NADP, a competitive inhibitor of malic enzyme with respect to NADP. inactivate the enzyme in mild conditions.
• 2.2. The inactivation is due to the modification of an essential lysine residue.
• 3.3. Two molecules of reagent were found to be incorporated into the enzyme tetramer after extensive modification.
• 4.4. Complete protection of malic enzyme from the oxidized NADP inactivation was afforded by NADP and its analogues.
• 5.5. The modified enzyme showed increased apparent Michaelis constant for the nucleotide coenzymes but the maximum velocity was decreased.
• 6.6. The binding between the modified enzyme and NADPH was impaired.

     

The measurement of glutamate dehydrogenase activity in Praunus flexuosus and its role in the regulation of ammonium excretion

• 1.1. A strong correlation (r² = 0.916) was found between glutamate dehydrogenase (GDH) activity and ammonium excretion in Praunus flexuosus suggesting a key regulatory role for this enzyme.
• 2.2. The high level of GDH activity found in this mysid was sufficient to account for all the ammonium excreted.
• 3.3. Activator-inhibitor studies imply that GDH may regulate energy production and growth, in addition to ammonium excretion.
• 4.4. The GDH assay presented here appears useful as a technique for quantifying zooplankton ammonium excretion impacts in the world oceans.

     

Distribution of glutamate dehydrogenase in the intestine of the earthworm (Lumbricus terrestris), and some physiological implications

• 1.1. Intestines of fresh and dehydrated-starved L. terrestris were compared to tissue and anterior-posterior distribution of glutamate dehydrogenase (GDH) and other mitochondrial or cytosol dehydrogenases.
• 2.2. For any dehydrogenase, including GDH, practically all the activity was in the gut epithelium. This distribution of GDH supports Tillinghast (1967, 1968) as to the excretory route for ammonia.
• 3.3. While the distributions of the marker dehydrogenases were reasonably uniform along the intestine, the GDH activity was predominantly (80–90% of the total activity) in the last third of the mid-intestine, indicating a true physiological differentiation of the midgut tube. The GDH activity of the typhlosole was about two times the activity in the peripheral epithelium. The GDH distribution was independent of the physiological state of the worm.
• 4.4. From the distribution of GDH it follows that the mid-intestine, immediately before the hindgut, is the main region both for amino acid uptake and catabolism. As regards amino acids, it typifies the primitive digestive tube by having both the absorptive and the liver functions.

     

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.

     

NADPH/NADP ratio could regulate the glyoxylate cycle in Tetrahymena pyriformis

• 1.1. The glyoxylic acid cycle pathway could be regulated through the modulation of the isocitrate dehydrogenase-NADP activity. This enzyme is inhibited by NADPH.
• 2.2. The effect on the glyoxylate cycle flux of variations in the rate of the NADPH-consuming pathways has been studied.
• 3.3. Increase in the rate of NADPH-consuming activity by addition of H₂O₂ produces inhibition of the glyoxylate cycle and decrease in the NADPH/NADP ratio.
• 4.4. These results suggest that the glyoxylate flux in Tetrahymena could be modulated by regulation of NADP-dependent isocitrate dehydrogenase by the NADPH/NADP ratio.

     

Purification and some properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium

• 1.1. A NAD⁺-dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium.
• 2.2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD⁺-dependent oxidative deamination of l-glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na⁺ than with K⁺ in the amination reaction while the reverse is true in the deamination reaction.
• 3.3. The apparent K_m values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l-glutamate, 4.0 mM; NAD⁺, 0.30 mM.
• 4.4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l-amino acids.

     

Effect of aminooxyacetate and α-ketoglutarate on glutamate deamination by rat kidney mitochondria

• 1.1. Glutamate dehydrogenase flux by rat kidney mitochondria incubated with 1 mM glutamine plus 2–3 mM glutamate was stimulated by aminooxyacetate. This effect was inhibited by α-ketoglutarate.
• 2.2. Studies with intact mitochondria and mitochondrial sonicates revealed a linear inverse relationship between glutamate deamination and α-ketoglutarate levels.
• 3.3. The data revealed that α-ketoglutarate is a competitive inhibitor of glutamate dehydrogenase with an apparent K_i of 0.6mM.
• 4.4. The data suggest that aminooxyacetate stimulates glutamate deamination by a mechanism mediated by α-ketoglutarate.

     

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.

     

Partial purification of rat liver cytoplasmic acetyl-CoA synthetase; Characterization of some properties

• 1.1. Rat liver cytoplasmic acetyl-CoA synthetase was partially purified (purification factor = 23, yield = 30%).
• 2.2. The apparent K_ms for acetate, coenzyme A, ATP and MgCl₂ were determined and found to be 52.5 μM, 50.5 μM, 570 μM and 1.5 mM, respectively.
• 3.3. The partially-purified enzyme showed a low affinity for short-chain carbon substrates other than acetate.
• 4.4. The properties of the partially-purified enzyme were compared with those of enzymes from other sources.

     

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.

     

The anaerobic metabolism of malate of Saccharomyces bailii and the partial purification and characterization of malic enzyme

1. The main pathway of the anaerobic metabolism of l-malate in Saccharomyces bailii is catalyzed by a l-malic enzyme.
2. The enzyme was purified more than 300-fold. During the purification procedure fumarase and pyruvate decarboxylase were removed completely, and malate dehydrogenase and oxalacetate decarboxylase were removed to a very large extent.
3. Manganese ions are not required for the reaction of malic enzyme of Saccharomyces bailii, but the activity of the enzyme is increased by manganese.
4. The reaction of l-malic enzyme proceeds with the coenzymes NAD and (to a lesser extent) NADP.
5. The K _m-values of the malic enzyme of Saccharomyces bailii were 10 mM for l-malate and 0.1 mM for NAD.
6. A model based on the activity and substrate affinity of malic enzyme, the intracellular concentration of malate and phosphate, and its action on fumarase, is proposed to explain the complete anaerobic degradation of malate in Saccharomyces bailii as compared with the partial decomposition of malate in Saccharomyces cerevisiae.

     

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.

     

In vitro substrate utilization for lipid synthesis in liver explants from hyperthyroid chickens

• 1.1. Indian River male broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 18, 24 and 30% protein + 0 or 1 mg triiodothyronine (T₃)/kg of diet to study energetic costs of lipogenesis and the use of various substrates for in vitro lipogenesis.
• 2.2. De novo lipid and CO₂ production were determined in the presence of [1-¹⁴C]pyruvate, [2-¹⁴q]pyruvate, [3-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine.
• 3.3. Oxygen consumption was determined in mitochondrial preparations to estimate the energetic costs in expiants synthesizing lipid.
• 4.4. Radiolabeled CO₂ derived from [1-¹⁴C]pyruvate was used as an estimate of coenzyme A availability in liver expiants. Lipids derived from [2-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine estimate relative substrate efficiency.
• 5.5. Labeled CO₂ production from [1-¹⁴C]pyruvate was greatest in that group fed a 12% protein diet and least in the group fed a 30% protein diet.
• 6.6. In addition, T₃ increased CO₂ production from [1-¹⁴C]pyruvate.
• 7.7. The production of ¹⁴CO₂ from the second carbon of pyruvate or acetate was increased by T₃.
• 8.8. The low-protein diet (12% protein) increased (P <0.05) lipogenesis.
• 9.9. Adding T₃ to the diets decreased carbon flux into lipid from all substrates, but increased CO₂ production from all substrates without changing stage 3 and 4 respiration rates in mitochondrial preparations.
• 10.10. These observations imply that coenzyme A availability may have regulated de novo lipogenesis in the present study.
• 11.11. It was also concluded that previously noted effects of T₃ on intermediary metabolism may involve metabolic pathways that do not involve changes in mitochondrial function.

     

Locust vitellogenin receptor: an acidic glycoprotein with N- and O-linked oligosaccharides

• 1.1. The locust vitellogenin (VTG) receptor which is embedded in oocyte plasma membranes is a glycoprotein.
• 2.2. With various lectins oligosaccharide units have been identified, among them neuraminic acid linked to Gal or GalNAc, mannose chains, Gal linked to GalNAc or GlcNAc and fucose linked to GlcNAc.
• 3.3. With specific enzymes it could be shown that mannose and most other oligosaccharides are O-linked while others like fucose are N-linked.
• 4.4. Enzymatic removal of all O-linked carbohydrates resulted in a drop of the molecular mass of the receptor protein from 200,000 to 110,000.
• 5.5. A total of N- and O-linked oligosaccharides of 54% was calculated.
• 6.6. The isoelectric point of the receptor was found to be at pH 3.4 increasing slightly after removal of neuraminic acid.
• 7.7. Removal of neuraminic acids destroyed the binding ability for VTG.

     

Metabolic and functional characteristics of erythrocytes from Glycera and Noetia

• 1.1. Annelid and molluscan red blood cells (RBC) may de differentiated metabolically from vertebrate RBC by their increased permeability to substrate, their magnitude of amino acid catabolism and their higher aerobic metabolism.
• 2.2. At 22°C, Glycera and Noetia RBC oxidize glucose and glutamate to CO₂ without accumulation of either d- or l-lactate. By comparison, the oxidation of glutamate by rat and chicken RBC is negligible at this temperature despite its incorporation into the cells.
• 3.3. At 37°C, chicken RBC oxidize glutamate at a rate 4 times greater than at 22°C, with oxygen uptake still lower than that in Noetia RBC at 32°C. At 37°C, rat RBC do not increase their oxidation of glutamate above that at 22°C, but oxygen uptake increases to slightly more than half that of chicken RBC.
• 4.4. Our finclings indicate that RBC of these two invertebrate species have both a higher aerobic metabolism and lower anerobic capacity than vertebrate RBC.
• 5.5. Moreover, the annelid and molluscan RBC have a relatively lower activity of the pentose phosphate (PPO₄) pathway than vertebrate RBC, as evidenced by their higher thermal sensitivity of oxygen uptake and their higher *C₁O₂/*C₆O₂ isotope ratio.

     

Biosynthetic pathways of Vibrio succinogenes growing with fumarate as terminal electron acceptor and sole carbon source

1. With fumarate as the terminal electron acceptor and either H₂ or formate as donor, Vibrio succinogenes could grow anaerobically in a mineral medium using fumarate as the sole carbon source. Both the growth rate and the cell yield were increased when glutamate was also present in the medium.
2. Glutamate was incorporated only into the amino acids of the glutamate family (glutamate, glutamine, proline and arginine) of the protein. The residual cell constituents were synthesized from fumarate.
3. Pyruvate and phosphoenolpyruvate, as the central intermediates of most of the cell constituents, were formed through the action of malic enzyme and phosphoenolpyruvate synthetase. Fructose-1,6-bisphosphate aldolase was present in the bacterium suggesting that this enzyme is involved in carbohydrate synthesis.
4. In the absence of added glutamate the amino acids of the glutamate family were synthesized from fumarate via citrate. The enzymes involved in glutamate synthesis were present.
5. During growth in the presence of glutamate, net reducing equivalents were needed for cell synthesis. Glutamate and not H₂ or formate was used as the source of these reducing equivalents. For this purpose part of the glutamate was oxidized to yield succinate and CO₂.
6. The α-ketoglutarate dehydrogenase involved in this reaction was found to use ferredoxin as the electron acceptor. The ferredoxin of the bacterium was reoxidized by means of a NADP-ferredoxin oxidoreductase. Enzymes catalyzing the reduction of NAD, NADP or ferredoxin by H₂ or formate were not detected in the bacterium.

     

Inhibition of the nad-linked glutamate dehydrogenase from Trypanosoma cruzi by sulfhydryl reagents

• 1.1. The NAD-linked glutamate dehydrogenase (EC 1.4.1.2) partially purified from epimastigotes of Trypanosoma cruzi was strongly inhibited by the sulfhydryl reagents fluorescein mercuric acetate (FMA), p-chloromercuribenzoate (p-CMB), 5,5′ dithiobis (2-nitrobenzoate) (DTNB), N-ethylmaleimide (NEM), o-iodosobenzoate (IBz) and iodoacetamide (IAm).
• 2.2. The [I]₅₀ values (concentration of inhibitor for 50% inhibition) were 0.12, 1, 20, 80 μM, 1.2 and 25 mM, respectively, and the inhibition was nearly complete. Iodoacetate was practically ineffective.
• 3.3. The inhibition by p-CMB or FMA, and to some extent that by DTNB, but not that by NEM or IBz, could be partially reversed by addition of β-mercaptoethanol.
• 4.4. The enzyme partially modified by preincubation with p-CMB or IBz presented the same apparent K_m values for α-oxoglutarate, NADH and NH₄Cl, with a decreased apparent V_max.
• 5.5. The results suggest that one or more sulfhydryl groups, at or near the active site, are required for the activity of this glutamate dehydrogenase, which seems to be the most sensitive to thiol reagents among the similar enzymes studied so far.

     

Role of glutamate dehydrogenase reaction in the control of citrate pool in yeast

• 1.1. Role of NADP-glutamate dehydrogenase in the depletion of citrate was analyzed using permeabilized yeast cells.
• 2.2. Citrate was converted to 2-oxoglutarate, which was then metabolized to glutamate by NADP-glutamate dehydrogenase in the presence of ammonium ion.
• 3.3. Formation of 2-oxoglutarate plus glutamate was in good agreement with the concentration of citrate decreased. Glutamate formation can be a good indicator of the depletion of citrate, because 70% of the citrate decreased was converted to glutamate.
• 4.4. Glycolytic activity was closely correlated with the decrease in citrate under the in situ conditions.
• 5.5. NADP-glutamate dehydrogenase increased in anaerobically grown yeast cells.
• 6.6. An effective depletion of citrate by increased synthesis of NADP-glutamate dehydrogenase can explain the lowered mechanism of citrate causing glycolytic stimulation under the anaerobic growth conditions of yeast.