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Developmental changes of two different isocitrate dehydrogenases, NAD+-linked (EC 1.1.1.41) and NADP+-linked (EC 1.1.1.42) enzymes, were determined in various rat tissues. In most adult tissues, either cytoplasmic or mitochondrial NADP+-linked enzyme showed the highest activity and NAD+-linked enzyme showed the lowest. Whereas, brain NAD+-linked enzyme activity predominated over both cytoplasmic and mitochondrial NADP+-linked enzyme activities, and furthermore it was the highest in all tissues examined. In adult brain, the higher activity was observed in the order of NAD+-linked, mitochondrial NADP+-linked and cytoplasmic NADP+-linked enzymes. However, brain NAD+-linked enzyme in newborn rats showed very low activity and cytoplasmic NADP+-linked enzyme activity was higher in the newborn stage than in the adult stage. Drastic changes of the brain isocitrate dehydrogenase activities occurred between 5 and 20 days after birth. During this period, cytoplasmic NADP+-linked enzyme decreased, mitochondrial NADP+-linked enzyme maintained, and NAD+-linked enzyme increased the activity. NAD+-linked enzyme activity in liver and kidney remained at a low level during development.

After establishing conditions which stabilize isocitrate lyase (EC 4.1.3.1) in cell-free preparations of Neurospora crassa , this enzyme was purified to homogeneity. The kinetic properties of the enzyme were studied in an effort to learn about the regulation of the enzyme in vivo, the mechanism of action and the properties of the active site. The enzyme was found to be inhibited by its products succinate and glyoxylate, and by phosphoenolpyruvate, fumarate, malate and fructose 1,6-bis-phosphate. The studies on the inhibition of the forward reaction by the enzyme products, glyoxylate and succinate, and studies of the kinetics of the back reaction showed that the enzyme functioned in a uni—bi reaction mechanism with succinate leaving before glyoxylate in the cleavage reaction. The inhibitions of the enzyme by maleate and the substrate analog homoisocitrate were examined, and the results shed light on the structure of the active site. In further studies, isocitrate lyase from both N. crassa and Pseudomonas indigofera was found to be competitively inhibited by the inorganic anions SO 4 2− , HPO 4 2− , Cl − and NO 3 − . The results obtained suggest that the isocitrate lyases from N. crassa and P. indigofera may have a common evolutionary background.

The mitochondrial fraction of rat liver homogenate catalyzed the conversion of cholesterol into 5-cholestene-3β,25-diol and 5-cholestene-3β,26-diol in the presence of an NADPH-generating system and oxygen. 5-Cholestene-3β,26-diol was the predominant product. 26-Hydroxylation of cholesterol was much more efficient with the NADPH-generating system than with NADPH alone, and it was shown that the isocitrate in the generating system was responsible for most of the stimulation. Addition of Mg2+ increased both the NADPH- and the isocitrate-dependent 26-hydroxylation, and at high concentrations of Mg2+ the 26-hydroxylation in the presence of NADPH was about the same as in the presence of isocitrate. Addition of Ca2+ increased NADPH-dependent but not isocitrate-dependent 26-hydroxylation. NADH and NADH-generating systems could not replace NADPH or the NADPH-generating system. However, NADH together with Mg2+ and ATP stimulated the reaction efficiently, probably due to energy-dependent intramitochondrial transhydrogenation. Citrate and isocitrate were found to stimulate mitochondrial 26-hydroxylation of cholesterol more efficiently than other citric acid cycle intermediates regardless of whether Mg2+ was present or absent. Mitochondrial 26-hydroxylation was inhibited by p-hydroxymercuribenzoate. Rotenone, potassium cyanide, and dinitrophenol had little or no effect on isocitrate-dependent 26-hydroxylation. Hg2+, Zn2+, Cu2+, and Fe3+ in 0.1 mm concentration inhibited the reaction strongly. Experiments with sonically disrupted mitochondria supported the contention that the mitochondrial 26-hydroxylase was mainly bound to the inner membranes. Experiments with 18O2 and 2H2O showed that in 25- as well as 26-hydroxylation of cholesterol the oxygen incorporated is derived from molecular oxygen.

The subunit structure and molecular weight of Escherichia coli NADP+-specific isocitrate dehydrogenase has been determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate at pH 7.0 and in 8 M urea at pH 2.9. The results obtained indicate that the enzyme, which exists as a catalytically active dimer, is composed of two subunits each with a molecular weight of approximately 42 000. Cross-linking with dimethylsuberimidate, followed by electrophoresis in sodium dodecylsulfate gives, in addition to a band of mol. wt 43 000, a second protein band of approximately 83 000. A single NH2-terminal amino acid, methionine, was obtained following dansylation of the performic acid oxidized protein suggesting identity of the subunits. The isoelectric point of the E. coli isocitrate dehydrogenase was found to be pH 5.0.

The regulatory properties of the NAD-specific isocitrate dehydrogenase (threo- d s-isocitrate:NAD oxidoreductase (decarboxylating), EC 1.1.1.41) from Blastocladiella emersonii have been studied with steady state kinetic methods. The enzyme was found to catalyze the oxidative decarboxylation of isocitrate as well as the reversed reaction. The results show that the enzyme is extremely sensitive to the buffer concentration in the assay mixture. This inhibition is reversed by the activators, AMP and citrate and is competitive with regard to isocitrate. AMP reduces K m for the substrates but has no effect on V . The enzyme exhibits positive homotropic cooperativity towards isocitrate both in the absence and presence of activators, while the cooperativity towards NAD disappears in the presence of activators as well as high isocitrate concentrations. The activation by AMP occurs according to normal Michaelis Menten kinetics. AMP increases the pH optimum of the reaction. The experimental results are consistent with the possibility that the enzyme contains two binding sites for isocitrate, a catalytic and a regulatory site. Binding of citrate as well as AMP mimics the effect of binding of isocitrate to the regulatory site.

Peroxisomes isolated on sucrose density gradients from homogenates of rat, chicken, or dog livers and rat kidney contained NAD + :α-glycerol phosphate dehydrogenase. Since the amount of sucrose in the peroxisomal fraction inhibited the enzyme activity about 70%, it was necessary to remove the sucrose by dialysis. About 8.4% of the total dehydrogenase of rat livers was in the surviving intact peroxisomes after homogenation. If corrected for particle breakage, this represented approximately 21% of the total activity. About 9.5% of the total enzyme was isolated in rat kidney peroxisomes, and because of severe particle rupture may represent over half of the total activity. No glycerol phosphate dehydrogenase was found in spinach leaf peroxisomes. A specific activity of 326 nmoles min −1 mg −1 protein in the rat liver peroxisomal fraction was at least twice that in the cytoplasm. NAD + :α-glycerol phosphate dehydrogenase was also present in a membrane fraction which was not identified, but none was in the mitochondria. The liver peroxisomal and cytoplasmic NAD + :α-glycerol phosphate dehydrogenase moved similarly on polyacrylamide gels and each resolved into two adjacent bands. Malate dehydrogenase was not found in peroxisomes from liver and kidney of rats and pigs, but 1–2% of the total particulate malate dehydrogenase was present in the peroxisomal area of the gradient from dog livers. However, this malate dehydrogenase in dog peroxisomal fractions did not exactly coincide with the peroxisomal marker, catalase. Malate dehydrogenase in dog liver mitochondria and in the peroxisomal fraction had similar pH optima and K m values and migrated similarly to the anode at pH 6.5 on starch gels as a major and a minor band. The cytoplasmic malate dehydrogenase had a different pH optimum and K m value and resolved into five different isoenzymes by electrophoresis. It is concluded that NAD + :α-glycerol phosphate dehydrogenase is in peroxisomes of liver and kidney, whereas malate dehydrogenase, present in peroxisomes of plants, is apparently absent in animal peroxisomes.

Isocitrate dehydrogenase (NADP + -dependent) and isocitrate lyase compete for substrate in peroxisomes (microbodies) in the ciliated protozoan Tetrahymena pyriformis . Thus, some of the factors that might regulate the metabolism of isocitrate in Tetrahymena peroxisomes were studied. The dehydrogenase has a much greater affinity for isocitrate than does the lyase, but is subject to a concerted feedback inhibition by oxalacetate and glyoxylate. Concentrations of 25 μ m of each cause almost complete inhibition. The degree of inhibition is greatly reduced unless NADP is present during a brief preincubation of the enzyme with the inhibitors. Either glyoxylate or oxalacetate alone is also capable of inhibiting the enzyme, although higher concentrations are required. In this case, there is no inhibition unless inhibitor and enzyme are preincubated in the presence of NADP. Neither the concerted inhibition nor that due to oxalacetate seems to be competitive with isocitrate. Properties of the enzyme from the soluble fraction of the cell were identical to those of the peroxisomal enzyme. Enzyme from mouse liver exhibited most of the properties observed with the Tetrahymena enzyme. The concerted inhibition of isocitrate dehydrogenase may be the means by which isocitrate in the peroxisome is shunted through the glyoxylate bypass.

A triphosphopyridine nucleotide-specific isocitrate dehydrogenase from Salmonella typhimurium has been shown to be inhibited by purine nucleoside di- and triphosphates. Other nucleotides do not have the same capacity to inhibit the reaction, and the presence of both a purine nucleus and a pyrophosphate moiety seems to be necessary. The nucleotides appear to have a dual action. At low concentration they are competitive inhibitors of TPN+, and at higher concentration they are primarily competitive inhibitors of isocitrate. Evaluation of kinetic data indicates a distinct difference in mechanism of inhibition between ATP and sodium pyrophosphate with respect to the coenzyme. Competition with TPN+ appears to be a function of the purine nucleus, while that with isocitrate probably is due to chelation of Mn++ by the pyrophosphate grouping of the nucleotide.

A polarographic technique suitable for kinetic studies of either isolated or bound pyridine nucleotide-linked dehydrogenases was used to investigate the isocitrate dehydrogenases of yeast mitochondria. Intact mitochondria were found to contain both triphosphopyridine nucleotide- and diphosphopyridine nucleotide-linked isocitrate dehydrogenases with properties very similar to those which have been reported for the isolated dehydrogenases. The DPN-linked enzyme in situ exhibits a nonclassical, sigmoid substrate against activity relationship with respect to isocitrate and is activated by adenosine 5'-monophosphate. The TPN-linked enzyme in situ exhibits a hyperbolic substrate against activity relationship which is unaffected by AMP. The DPN-linked dehydrogenase also shows another non-classical increase in activity that occurs in response to high isocitrate concentrations or to heat treatment. This activation is characteristic only of untreated, intact mitochondria and may be secondary to an alteration of the mitochondria themselves.

As a continuation of work on the regulatory properties of plant enzymes, NAD isocitrate dehydrogenase (L s -isocitrate:NAD oxidoreductase (decarboxylating), EC 1.1.1.41) from swede ( Brassica napus L) storage tissue has been studied. It is shown that citrate stimulates the enzyme at low isocitrate concentrations and that the NAD:NADH ratio is important in regulating the enzyme. AMP and Pi do not affect the enzyme but ATP is very inhibitory possibly due to magnesium chelation. Previously it was shown that phosphofructokinase has a role in the regulation of glycolysis in plants (1, 2). The NAD isocitrate dehydrogenase from yeast has very complex regulatory properties (3). Is has been suggested that the tricarboxylic acid cycle is controlled by this enzyme (4). The same enzyme from Neurospora crassa is stimulated by citrate and this has been termed precursor activation (5). In all the cases studied the enzyme is greatly stimulated by low concentrations of either AMP of ADP. The enzyme exhibits these regulatory properties while still attached to the mitochondrion (6). NAD isocitrate dehydrogenase has been studied in swede (7) and pea seedlings (8) but no regulatory properties were described.

Isocitrate dehydrogenase (IDH) exists in mammalian tissues as three enzymes, one specific for NAD and two specific for NADP. The two NADP isozymes differ in electrophoretic mobility, immunological characteristics, and tissue and subcellular location. ONe is found mainly in the mitochondrial fraction and one in the supernatant fraction of tissue homogenates. Two allelic forms of the supernatant NADP isocitrate dehydrogenase have been described in inbred strains of mice. In a heterozygote containing both alleles, three forms of the supernatant NADP-IDH are generated in a ratio of 1:2:1. This implies that the supernatant enzyme is a dimer. The mitochondrial form is not affected by the gene, indicating separate genetic control. Liver mitochondria contain both the supernatant and the mitochondrial isozymes. Thus at least one protein found in the mitochondria is encoded in a nuclear gene.

Spinach chloroplast glyceraldehyde phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate: NADP oxidoreductase, phosphorylating; EC 1.2.1.13) is an equilibrium mixture of aggregates of a basic protomer (Mr about 145,000) and is active with both NADP and NAD. The enzyme is primarily “tetrameric” (Mr about 600,000), although minor amounts of smaller and larger oligomers are also found. Gel chromatography in buffer containing 30 μ m NADP results in depolymerization of the enzyme, mainly to protomers. NAD does not dissociate and counteracts this effect of NADP. The apparent Km values of the protomers are 7 μ m (NADP) and 8 μ m (NAD). The aggregates with a Mr > 106 have properties similar to the protomers. The tetramer as first isolated has higher Mm values for NADP (380 μ m ) and NAD (48 μ m ), but its apparent affinity for NADP is further decreased by repeated gel filtrations in buffer or by a single one in buffer containing NAD. Such preparations display nonlinear kinetics when NADP is the varied substrate and have a Km (NADP) of about 1.5–3.3 μ m . All these effects are reversible. V values are apparently the same in all enzyme forms and the V ( NADP ) V ( NAD ) ratio always approaches 2. Since, however, the enzyme is presumably dissociated by the NADP concentrations required for a “saturating” assay, the significance of V (NADP) seems questionable.

Acetate-grown Bacillus megaterium KM possessed high isocitrate lyase and malate synthase activity as compared to glucose-grown cells. Chloramphenicol prevented the increase in isocitrate lyase activity when cells were transferred from glucose to acetate media, indicating that such an increase in activity was probably due to de novo protein synthesis.The affinity of the substrate, isocitrate, was greater for isocitrate dehydrogenase than for isocitrate lyase. Phosphoenolpyruvate was found to inhibit isocitrate lyase non-competitively. The concerted action of glyoxylate and oxaloacetate was capable of inhibiting isocitrate dehydrogenase. The role such factors play in the balancing of the tricarboxylic acid cycle and the glyoxylate pathway in the microorganism is considered.

Isocitrate dehydrogenase from Azotobacter vinelandii transfers hydrogen from threo - d 8 -isocitrate to the A side of TPN + . The decarboxylation of threo d 8 isocitrate to 2-ketoglutarate occurs with retention of configuration. The enzyme catalyzes the labilization of tritium from [3-T]2-ketoglutarate in the presence of Mg 2+ and TPNH which suggests an ordered addition of substrates in the reductive carboxylation of 2-ketoglutarate to isocitrate.

An NAD+-dependent isocitrate dehydrogenase (threo- d s-isocitrate:NAD+ oxidoreductase (decarboxylating), EC 1.1.1.41) has been purified more than 500-fold from porcine hearts. Disc gel electrophoresis reveals that protein with NAD+-specific isocitrate dehydrogenase activity accounts for 80% of the total protein; one major and two minor activity bands are observed. As compared to the NADP+-specific isocitrate dehydrogenase isolated from the same species and organ, the NAD+-dependent enzyme differs in molecular weight, isoelectric point and reaction to antisera prepared against the purified NADP+ enzyme.

Interaction of NADPH and NADP+ with the oxidized (E) and 2-electron reduced (F) states of yeast glutathione reductase was studied at pH 7.6 and 25°. It is concluded that either NADPH or NADP+ can bind to both E and F. Spectrophotometric evidence is presented for formation of an F-NADPH complex with a stoichiometry of one NADPH per enzyme-bound FAD. The dissociation constant for this complex is 2.1 µm and its formation is complete in about 15 msec. Two previously unreported spectral species, considered to represent complexes between F and NADP+, result on anaerobic reduction of E by NADPH in the presence of high NADP+ concentrations. The more rapidly formed F-NADP+ complex slowly undergoes further spectral change to the second, "stable" species, F-NADPs+. Sufficiently high NADPH concentrations prevent formation of either F-NADP+ or F-NADPs+, suggesting that NADPH and NADP+ compete for a common site, probably the active site. The affinity of that site is greater for NADPH than for NADP+. Competitive inhibition by NADP+ of reduction of enzyme-bound FAD by NADH (studied by stopped flow methods) and of NADH-dependent GSSG reduction (studied by steady state kinetics) provide kinetic evidence for an E-NADP+ complex with a dissociation constant of 70 µm.

A novel form of allosteric control in which NAD-specific glutamic dehydrogenases obtained from taxonomically related fungi are activated by NADP+, NADPH, and P-enolpyruvate has been elucidated. Citrate and isocitrate are equally effective as inhibitors of the enzymes. The enzymes are allosterically inhibited by their substrates, α-ketoglutarate and ammonia, in a highly cooperative manner. NADP+, NADPH, and P-enolpyruvate antagonize substrate inhibition. These activators also function as multivalent modulators in releasing the enzyme from inhibition by citrate. Control of the enzyme activity is unidirectionally oriented such that it is the reductive amination that is effectively controlled. The purified glutamic dehydrogenases have a molecular weight of 225,000 daltons. They are susceptible to inactivation by hydrodynamic shear in sucrose density gradients at alkaline pH levels. Inactivation by sedimentation can be prevented by bound activators. Inactivation and dissociation of the enzyme are related phenomena. Synthesis of the glutamic dehydrogenases can be repressed by addition of glucose to the growth medium. Glutamate acts as an inducer. NADP-specific isocitric dehydrogenase present in these fungi responds in the opposite manner to the metabolism of glucose and glutamate.

The intracellular distributions of a number of NAD+-specific and NADP+-specific dehydrogenases in the stems of etiolated pea seedlings and in other nonphotosynthetic plant tissues have been determined. The NADP+-specific enzymes were found to be located almost exclusively in the soluble fraction, whereas the NAD+-specific enzymes were predominantly mitochondrial. There was no appreciable NADPH → NAD+ transhydrogenase activity in any of the isolated fractions. On the other hand, active homotranshydrogenases of the type NADH → NAD+ and NADPH → NADP+ were detected; the former is predominantly mitochondrial, while the latter is found in both the microsomal and soluble fractions. The possibility that these transhydrogenase activities are due to enzymes which normally catalyze other reactions is considered. The results support the view that in plant tissues, as in animals, there is a “division of labor” between NAD and NADP.

Product inhibition studies with Rhodopseudomonas spheriodes NADP + specific isocitrate dehydrogenase indicate that the enzyme mechanism involves the ordered addition of the substrates NADP + and threo- d s -isocitrate and the ordered release of products CO 2 (HCO s − ), 2-ketoglutarate, and NADPH. In addition, the presence of a ternary complex consisting of enzyme, NADP + , and 2-ketoglutarate is indicated. Binding studies with radioactive substrates support the kinetically derived mechanism. The Rhodopseudomonas enzyme is dimeric and contains but a single active site. Different combinations of substrate were ineffective in causing gross changes in molecular structure as monitored by gel filtration techniques. A comparison of the amino acid composition of this enzyme with the bacterial enzyme from Azotobacter vinelandii indicate very significant differences in the amino acid compositions.

Oxygen uptake, reduction of added pyridine nucleotides and enzyme activities concerned with isocitrate oxidation show that aged mitochondria from both neonatal and adult rat brain oxidize isocitrate by two separate and independent pathways, one mediated by NAD and NADH 2 -oxidase system and the other by NADP and NADPH 2 -oxidase system. The neonatal and adult brain mitochondria contain equal concentrations of NAD-isocitrate dehydrogenase ( l S -isocitrate: NAD oxidoreductase (decarboxylating), EC 1.1.1.41) and NADH 2 -cytochrome c reductase (NADH 2 : cytochrome c oxidoreductase, EC 1.6.2.1) activities. However, the neonatal mitochondria have a much greater NADP-isocitrate dehydrogenase ( l S -isocitrate: NADP oxidoreductase (decarboxylating), EC 1.1.1.42) activity and much lower NADPH 2 -cytochrome c reductase (NADPH 2 : cytochrome c oxidoreductase, EC 1.6.2.3) activity than the adult. Oxidation of citrate occurred at a lower rate than isocitrate oxidation in intact mitochondria from both neonate and adult brains. But in aged mitochondria from the adult, citrate was oxidized at a higher rate than isocitrate indicating an alternate pathway for citrate oxidation in this preparation in addition to the tricarboxylic acid cycle. Intact mitochondria from adult rat brain oxidized citrate and isocitrate with a P:O ratio of 2. This ratio was increased by added NAD and decreased by added NADP. Mitochondria aged in phosphate buffer failed to oxidize either citrate or isocitrate unless pyridine nucleotides were supplemented. Addition of either NAD or NADP restored oxidation completely, but the oxidative phosphorylation did not exceed a ratio of 1.

Highly purified NADP+-linked isocitrate dehydrogenase was X-irradiated in dilute aqueous solution and proved to be very radiosensitive, being inactivated with a G value of 0.5. The catalytic functions of the enzyme were destroyed at the same rate. However, unlike most allosteric enzymes, the allosteric properties were not more radiosensitive than the catalytic properties. X-ray inactivation was associated with the destruction of seven to eight sulfhydryl groups. The first four sulfhydryls destroyed were extremely radiosensitive, having G values for destruction of almost 2. Comparison with several reagents known to attack sulfhydryl groups suggested that sulfhydryl destruction was sufficient to account for the X-ray response of the enzyme. Destruction of the single essential methionine did not seem significant in the inactivation. X-ray inactivation was also associated with increases in the Km values for isocitrate and manganese.

The increased isocitrate consumption of isolated rat liver mitochondria brought about by the addition of L-malate is quantitatively accounted for by increases in the production of both α-ketoglutarate and citrate. The effect on citrate production is still observed under conditions such that the oxidation of isocitrate to α-ketoglutarate is completely inhibited. The specificity of the effect has been investigated, and it is shown that a number of dicarboxylic acids can stimulate isocitrate metabolism in a similar fashion. Marked stereospecificity is indicated by the fact that among the tartrate stereoisomers only the meso form is active.

The NADP-dependent isocitrate dehydrogenase from pig liver soluble fraction was purified over 500-fold with an overall yield of 25%. The purified enzyme, which is homogeneous by all the usual criteria, has a molecular weight of about 75000 and is composed of two identical subunits. This has been demonstrated by ultracentrifugation, fluorescence titration and peptide `fingerprinting'. The maximal turnover number, extinction coefficients at 280nm and 260nm and amino acid analysis are described.

A mitochondrial fraction was isolated from chick epiphyseal cartilage using a Polytron. The oxidative phosphorylating properties and principle dehydrogenase activities of these mitochondria were studied. The highest rate of oxidation was noted when succinate was used as a substrate. Lesser levels of respiration were recorded with isocitrate in the presence of NADP. Succinate, isocitrate, oxoglutarate, malate and glycerol-3-phosphate dehydrogenases were found in chondrocyte mitochondria. Of these, succinate and NADP-linked isocitrate dehydrogenases were the most active. The results indicate that chondrocyte mitochondria have the enzymic capability to perform oxidative phosphorylation. The presence of NADP-linked isocitric dehydrogenase supports the view that this enzyme may play a key role in citrate formation.

Experiments designed to elucidate the nature of 17β-hydroxysteroid dehydrogenase from human red blood cells have shown that NADP+ activates and protects the enzyme, while also serving as substrate for the reaction. Enzyme activity was measured by the conversion of 17β-estradiol to estrone and by the production of NADPH with 17β-estradiol-3-sulfate as substrate. It appears that the reaction sequence is first, binding with NADP+ and second, binding with the steroid. The binding with NADP+ is essentially irreversible: the activated enzyme is completely protected against loss of activity by dilution. On dilution of the unactivated enzyme, much of the activity is lost. The bireactant rate equation of the sequential type has been restated for the case of activation by one of the reactants. Since it has been found that activation of enzyme is linear with NADP+ concentration, it follows that the Michaelis constant for the steroid substrate is independent of the concentration of NADP+ activating the enzyme. This is substantiated by the determination of the Michaelis constant for 17β-estradiol-3-sulfate from data on double-reciprocal plots of activated and unactivated enzyme with limiting amounts of steroid. The activating effect increases linearly up to a concentration of 1.2 × 10−5 m of NADP+ and then levels off. The activation is highly specific for NADP+; neither NAD+, ATP, NADPH, nicotinic acid, ncr nicotinamide prevent the loss of activity after storing the enzyme for 1 hr at 37 °C. The steroid substrate appears to interfere with the activation of NADP+.

The cation-complexing carboxylic-acid antibiotic X-537A, at concentrations far below that required for ionophorous activity, selectively inhibits the oxidation of glutamate and isocitrate by liver mitochondria in steady-state 3. The site of inhibition has been localized specifically at the reduction of NADP + . Glutamate and isocitrate dehydrogenases, the oxidation of NAD + -dependent substrates, pyridine nucleotide transhydrogenations, and the respiratory chain between NADH (or NADPH) and O 2 are unaffected by the antibiotic X-537A. Kinetic evidence, i.e., competition between chlorotetracycline (a fluorescence probe for membrane-bound bivalent cation) and X-537A, indicates that the NADP + -reducing, antibiotic-sensitive site is most probably associated with the inner mitochondrial membrane.

Bilirubin was found to be a potent inhibitor of NAD+-linked isocitrate dehydrogenase (threo-ds-isocitrate:NAD+ oxidoreductase (decarboxylating), EC 1.1.1.41). It inhibited the enzyme by decreasing the affinity of enzyme for isocitrate, while the maximum reaction velocity was not affected. Bilirubin (less than 10−6 M) showed a detectable inhibition. On the other hand, neither the mitochondrial nor the cytoplasmic NADP+ enzymes (threo-ds-isocitrate:NADP+ oxidoreductase ((decarboxylating), EC 1.1.1.42) were affected by bilirubin.

1. The NAD(P) transhydrogenase activity of the soluble fraction of sonicated rat liver mitochondrial preparations was greater than the NAD-linked isocitrate dehydrogenase activity, and the NAD-linked and NADP-linked isocitrate dehydrogenase activities were not additive. The NAD-linked isocitrate dehydrogenase activity was destroyed by an endogenous autolytic system or by added nucleotide pyrophosphatase, and was restored by a catalytic amount of NADP. 2. We concluded that the isocitrate dehydrogenase of rat liver mitochondria was exclusively NADP-specific, and that the oxoglutarate/isocitrate couple could therefore be used unequivocally as redox reactant for NADP in experiments designed to operate only the NAD(P) transhydrogenase (or loop 0) segment of the respiratory chain in intact mitochondria. 3. During oxidation of isocitrate by acetoacetate in intact, anaerobic, mitochondria via the rhein-sensitive, but rotenone- and arsenite-insensitive, NAD(P) transhydrogenase, measurements of the rates of carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive pH change in the presence of various oxoglutarate/isocitrate concentration ratios gave an →H+/2e− quotient of 1.94±0.12 for outward proton translocation by the NAD(P) transhydrogenase. 4. Measurements with a K+-sensitive electrode confirmed that the electrogenicity of the NAD(P) transhydrogenase reaction corresponded to the translocation of one positive charge per acid equivalent. 5. Sluggish reversal of the NAD(P) transhydrogenase reaction resulted in a significant inward proton translocation. 6. The possibility that isocitrate might normally be oxidized via loop 0 at a redox potential of −450mV, or even more negative, is discussed, and implies that a P/O quotient of 4 for isocitrate oxidation might be expected.

1. The factors capable of affecting the rate of isocitrate oxidation in intact mitochondria include the rate of isocitrate penetration, the activity of the NAD-specific and NADP-specific isocitrate dehydrogenases, the activity of the transhydrogenase acting from NADPH to NAD+, the rate of NADPH oxidation by the reductive synthesis of glutamate and the activity of the respiratory chain. A quantitative assessment of these factors was made in intact mitochondria. 2. The kinetic properties of the NAD-specific and NADP-specific isocitrate dehydrogenases extracted from rat liver mitochondria were examined. 3. The rate of isocitrate oxidation through the respiratory chain in mitochondria with coupled phosphorylation is approximately equal to the maximal of the NAD-specific isocitrate dehydrogenase but at least ten times as great as the transhydrogenase activity from NADPH to NAD+. 4. It is concluded that the energy-dependent inhibition of isocitrate oxidation by palmitoylcarnitine oxidation is due to an inhibition of the NAD-specific isocitrate dehydrogenase. 5. Kinetic studies of NAD-specific isocitrate dehydrogenase demonstrated that its activity could be inhibited by one or more of the following: an increased reduction of mitochondrial NAD, an increased phosphorylation of mitochondrial adenine nucleotides or a fall in the mitochondrial isocitrate concentration. 6. Uncoupling agents stimulate isocitrate oxidation by an extent equal to the associated stimulation of transhydrogenation from NADPH to NAD+. 7. A technique is described for continuously measuring with a carbon dioxide electrode the synthesis of glutamate from isocitrate and ammonia.

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora , isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.

Cell-free extract supernatant fluids of Pseudomonas aeruginosa were shown to lack malic dehydrogenase but possess a nicotinamide adenine dinucleotide (NAD)- or NAD phosphate (NADP)-dependent enzymatic activity, with properties suggesting a malic enzyme (malate + NAD (NADP) → pyruvate + reduced NAD (NADH) (reduced NADP [NADPH] + CO 2 ), in agreement with earlier findings. This was confirmed by determining the nature and stoichiometry of the reaction products. Differences in heat stability and partial purification of these activities demonstrated the existence of two malic enzymes, one specific for NAD and the other for NADP. Both enzymes require bivalent metal cations for activity, Mn 2+ being more effective than Mg 2+ . The NADP-dependent enzyme is activated by K + and low concentrations of NH 4 + . Both reactions are reversible, as shown by incubation with pyruvate, CO 2 , NADH, or NADPH and Mn 2+ . The molecular weights of the enzymes were estimated by gel filtration (270,000 for the NAD enzyme and 68,000 for the NADP enzyme) and by sucrose density gradient centrifugation (about 200,000 and 90,000, respectively).

Summary It has been found that a number of agents greatly affects the release by Triton-X-ioo of certain dehydrogenases from rabbit-brain mitochondria. The enzymes studied were DPN-dependent isocitrate dehydrogenase ( L s-isocitrate:DPN+ oxidoreductase (decarboxylating), EC 1.1.1.41) and TPN-dependent isocitrate dehydrogenase ( L s-isocitrate:TPN+ oxidoreductase (decarboxylating), EC 1.1.1.42), gluta-mate dehydrogenase ( L -glutamate:DPN+ (TPN+) oxidoreductase (deaminating), EC 1.4.1.3) and lipoamide dehydrogenase (DPNH:lipoamide oxidoreductase, EC 1.6.4.3). The effects of Triton X-100 on the morphology of the mitochondria were checked with the electron microscope. 1. The detergent was shown to disrupt the architecture of the mitochondria, leaving single and double membranes and amorphous material. 2. ATP, ADP, pyrophosphate, citrate and isocitrate enhanced release of the enzymes. DPN was less effective, and α-ketoglutarate, adenylic acid and adenosine were ineffective, as was EDTA. 3. K+ and Na+ (80 mM) enhanced release of both isocitrate dehydrogenases, but partially prevented release of glutamate dehydrogenase. Ca2+ and Mg2+ at 4–5 mM enhanced release of TPN-isocitrate dehydrogenase but inhibited that of DPN-isocitrate dehydrogenase and glutamate dehydrogenase. Mg2+ had little effect on the release of lipoamide dehydrogenase. 4. It is suggested that metal ions play a role in the binding of these four dehydrogenases to the mitochondria, but that the details of the attachments may be different in each case.

Pure isocitrate dehydrogenase from pig liver cytoplasm catalyses the reduction of oxaloacetate by NADPH at a rate comparable with that observed for the usual substrates. The products are NADP and D-malate, the ‘unatural’ isomer. High concentrations of magnesium (25 mM) are necessary for maximal activity, and the reaction is not appreciably reversible. These results are discussed in connection with the inhibition of the enzyme by mixtures of glyoxylate and oxaloacetate. The reduction is not thought to be of physiological importance.

Several isoenzymes of lactate, malate, and isocitrate dehydrogenases have been demonstrated in human skeletal muscle, heart, and liver. Single zones of activity were shown for glucose-6-phosphate and glutamate dehydrogenases. A zone possessing a marked reducing capacity towards nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate was visualized in the absence of any substrate, using the tetrazolium-coupled histochemical method. This zone represents the so-called "nothing dehydrogenase".

Publisher Summary The anaplerotie function of isocitrate lyase and malate synthase during microbial growth on acetate is well documented. Catalysis by these enzymes is also vital in the conversion of lipid reserves to carbohydrates, as found, for example, early in the germination of fatty plant seedlings. Catalysis by isocitrate lyase also provides glyoxylate for condensation with higher fatty acids during microbial growth on these compounds, for glycine synthesis in nematodes, and for oxalate accumulation in Oxalis. Microbial growth on acetate enhances formation of isocitrate lyase in contrast to tricarboxylic acid cycle intermediates, glucose, or complex media which markedly suppress the formation. A much more relaxed repressive control of isocitrate lyase exists in Pseudomonas indigofera. Enzymatic activity is measured by determining the amount of glyoxylate formed from threo- Ds - isocitrate in 10 minutes at 30°. A slight modification of a highly sensitive and relatively specific colorimetric assay for glyoxylate is employed.

Acetobacter suboxydans is an obligate aerobe for which an operative tricarboxylic acid cycle has not been demonstrated. Glutamate synthesis has been reported to occur by mechanisms other than those utilizing isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme not previously detected in this organism. We have recovered α-ketoglutarate and glutamate from a system containing citrate, nicotinamide adenine dinucleotide (NAD), a divalent cation, pyridoxal phosphate, an amino donor, and dialyzed, cell-free extract. Aconitase activity was readily detected in these extracts, but isocitrate dehydrogenase activity, measured by NAD reduction, was masked by a cyanide-resistant, particulate, reduced NAD oxidase. Isocitrate dehydrogenase activity could be demonstrated after centrifuging the extracts at 150,000 × g for 3 hr and treating the supernatant fluid with 2-heptyl-4-hydroxyquinoline N -oxide. It is concluded that A. suboxydans can utilize the conventional tricarboxylic acid cycle enzymes to convert citrate to α-ketoglutarate which can then undergo a transamination to glutamate.

Electrophoretically distinct forms of nicotinamide adenine dinucleotide phosphate-specific isocritrate dehydrogenase have been observed in extracts of Escherichia coli grown under different culture conditions. In glucose-grown cells, two distinct bands of isocitrate dehydrogenase activity were observed on polyacrylamide gels and have been completely resolved by employing ion-exchange chromatography. These multiple forms of the enzyme have been studied and their possible metabolic role is discussed.

A possible physiologic role for the concerted inhibition of a NADP+-specific isocitrate dehydrogenase by oxalacetate and glyoxylate is presented. The significance of this inhibition is also discussed with respect to the “induced fit” theory of enzyme-substrate interaction. However, since numerous structural analogues of these two compounds were without effect, it is believed that these two components are not acting as a “synthetic” substrate.

A recent claim in the literature (Moyle, J. and Mitchell, P. (1973) Biochem. J. 132, 571–585) that the NAD-dependent isocitrate oxidation observed in extracts of ratliver mitochondria proceeds entirely via the NADP-linked isocitrate dehydrogenase and nicotinamide nucleotide transhydrogenase, and that rat-liver mitochondria contain no NAD-linked isocitrate dehydrogenase, has been examined by using palmityl-CoA as a selective inhibitor of transhydrogenase and ADP as a selective activator of the NAD-linked isocitrate dehydrogenase. The results unambiguously demonstrate that the NAD-dependent oxidation of isocitrate observed under the conditions employed by Moyle and Mitchell proceeds predominantly via the NAD-linked isocitrate dehydrogenase. It is also shown that, by an unfortunate choice of assay conditions, these authors have considerably overestimated the rate of the transhydrogenase reaction.

Publisher Summary This chapter discusses NAD-specific isocitrate dehydrogenase from Neurospora crassa, serving a regulatory function in vivo, and belonging to a class of enzymes which have been termed “allosteric.” These enzymes are characterized by the presence of an allosteric site which normally binds effectors that may be totally unrelated to the substrate. The allosteric nature of isocitrate dehydrogenase is reflected in the marked deviations of the initial velocity data from normal Michaelis-Menten kinetics, when isocitrate is used as the variable substrate. The enzyme is activated at pH 7.6 by citrate, which binds to the allosteric site. The substrate, isocitrate, also activates the enzyme by binding to the allosteric site. 5’-AMP activates isocitrate dehydrogenase apparently by increasing the affinity of both the allosteric and active sites. This effect is specific; 5’-GMP, 5’-IMP, 5P-UMP, 5’-CMP, ADP, and ATP tested at a concentration of 1.6 mM do not substitute for AMP. At pH 6.5 where the allosteric site is inoperative, the activators citrate and isocitrate, and the inhibitors, a-ketoglutarate and glutamate, have no effect on enzyme activity.

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ oxido-reductase, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.44) of rat adipose tissue, were shown to react not only with NADP+ but also with NAD+. Different isoenzymes were involved in the NAD+-linked and NADP+-linked reactions. The intracellular distribution of the NAD+-linked isoenzymes differed from that of NADP+-linked isoenzymes.

The effect of oxidizable substrates, α-ketoglutarate and isocitrate on 11β-hydroxylation in guinea-pig adrenal mitochondria has been studied. These substrates supported the conversion of Compound S (17α,21-dihydroxy-pregnene-3,20-dione) into cortisol as well as exogenously added NADPH in Ca 2+ -swollen mitochondria. Progesterone was also hydroxylated at the C-11β position but not at the C-21 position. Microsomes, on the other hand, when NADPH was added, converted pregnenolone, progesterone and 17α-hydroxyprogesterone into Compound S, but showed no steroid 11β-hydroxylating activity. Evidence obtained in incubations carried out with α-ketoglutarate and isocitrate in the presence of 2,4-dinitrophenol, oligomycin, amytal and antimycin A indicate that α-ketoglutarate utilization for steroid 11β-hydroxylation is dependent on activity of the classical electron chain. This activity can be related to high energy intermediates possibly needed for NADPH reduction arising from NADH oxidation via the energy-requiring transhydrogenase reaction. These reactions do not appear necessary for isocitrate utilization and isocitrate oxidation probably gives rise to intramitochondrial NADPH reduction as a result of a NADP + -linked isocitrate dehydrogenase. Data obtained in oxygen uptake studies with an antimycin A blocked system supplied with α-ketoglutarate, are in accordance with this conclusion. The high-speed supernatant fraction (103000 × g ) could partially replace α-ketoglutarate, isocitrate or Ca 2+ + NADPH, indicating that it contains a factor(s) (the physiological substrate?) which brings about intramitochondrial NADPH.

The isocitrate dehydrogenases of rat liver were found extremely sensitive to inacvation by linoleic acid hydroperoxide. Lipid peroxide was much more effective than other peroxides and sulfhydryl reagents tested. Dehydrogenase activity of crude subcellular fractions was more sensitive than the activity of the purified enzyme. The inactivation was not catalyzed by heme compounds. Loss of enzyme activity was accompanied by a decrease in the SH content of the enzyme. Inactivation was facilitated at elevated pH. The binding of p -chloromercuribenzoate to the enzyme protected against peroxide. The SH groups of isocitrate dehydrogenase were more reactive with linoleic acid hydroperoxide than any SH groups so far tested. It was concluded that inactivation is probably due to SH destruction and that modification of an essential methionine residue is unlikely to be a contributory factor. The unique features of the inactivation of isocitrate dehydrogenase by lipid peroxide are discussed.

The mechanism of isocitrate oxidation by liver mitochondria has been studied by several investigators, in particular to determine whether substrate dehydrogenation occurs by way of the TPN-linked or the DPN-linked isocitrate dehydrogenases (for review see Plaut (1963)). Initial studies of DPN-linked isocitrate dehydrogenase from animal tissues demonstrated the activity in extracts of acetone powders of mitochondria from heart, pigeon breast muscle and kidney. The TPNand DPN-linked dehydrogenases were separated in the cases of heart, pigeon breast muscle (Plaut and Sung, 1954), and human placenta (Sung and Hsu, 1957). However, the reduction of DPN by isocitrate was very slow with preparations from liver mitochondria although relatively high activity for the corresponding TPN-linked enzyme was found. These experiments thus did not prove the occurrence of the DPN enzyme in liver. The finding that DPN-linked enzyme from heart is activated by ADP (Chen and Plaut, 1963) prompted reinvestigation of the levels of this enzyme in various tissues including liver (Goebell and Klingenberg, 1964; Stein et al., 1967). However, the measurement of the DPN enzyme at 340 rnp in the presence

Publisher Summary This chapter discusses the occurrence of the enzymes in microorganisms, plants, and animals as well as their distribution in animal tissues. The DPN-linked isocitrate dehydrogenase activity was first detected and separated from the corresponding TPN-linked enzyme in yeast. Intramitochondrial isocitrate oxidation is controlled by factors relating to the energy-linked electron transport system. The DPN-linked enzyme occurs in a number of other microorganisms, such as, Aspergillus niger, Neurospora crassa, Acetobacter suboxidans, Blastocladiella emersomi, and Thiobacillus thiooxidans. In contrast to the corresponding TPN enzyme, the DPN isocitrate dehydrogenases from heart and yeast do not catalyze the decarboxylation or reduction by reduced pyridine nucleotide of oxalosuccinate. In the presence of the heart enzyme, the hydrogen atom from the α -position of threo -D S -isocitrate is transferred to the α side of the nicotinamide ring of DPN. In the presence of relatively low concentrations of isocitrate and without ADP, the enzyme is maximally active in the rather narrow range of pH 6.5–6.9.

The kinetic properties of NAD+-linked isocitrate dehydrogenase of yeast mitochondria have been examined by a polarographic technique which permits measurement of the enzyme in situ. The results not only show a cooperative interaction of the bound enzyme with isocitrate which is responsible for a substrate activation of relatively low concentrations, but also another activation which occurs at relatively high concentrations. The first substrate activation can be displaced to very low concentrations of isocitrate by the presence of AMP and was previously shown to be a property of the isolated dehydrogenase. In contrast, the second activation is not affected by AMP and has not been noted for soluble preparations. Such activation is characterized by a downward deflection of the double-reciprocal plot as the curve approaches high substrate concentrations. This non-linearity is observed only with the dehydrogenase in intact mitochondria, and it decreases or disappears when the mitochondria are heated or treated with deoxycholate. The nature of this activation has been examined further, and the data suggest that it is not the result of a direct effect on the bound dehydrogenase, but that it arises secondarily as a result of processes which increase the availability of substrate to the enzyme within the mitochondrion.

Of the two NADP-linked isocitrate dehydrogenases in Acinetobacter lwoffi the higher-molecular-weight form, isoenzyme-II, is reversibly stimulated sixfold by low concentrations of glyoxylate or pyruvate. Kinetic results indicate that this stimulation of activity involves both an increase in Vmax. and a decrease in the apparent Km values for substrates, most markedly that for NADP+. Other changes brought about by glyoxylate or pyruvate include a shift in the pH optimum for activity and an increased stability to inactivation by heat or urea. Mixtures of glyoxylate plus oxaloacetate, known to inhibit isocitrate dehydrogenases from other organisms, produce inhibition of both A. lowffi isoenzymes, and do not reflect the stimulatory specificity of glyoxylate for isoenzyme-II. Isoenzyme-II is also stimulated by AMP and ADP, but the activation by glyoxylate or pyruvate is shown to be quite independent of the adenylate activation. Differential desensitization of the enzyme by urea to the two types of activator further supports the view that the enzyme possesses two distinct allosteric regulatory sites. The metabolic significance of the activations is discussed.