Characteristics of enzymes forming 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) in brain

The subcellular localization and character of the enzymes forming 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) were determined in rat brain. The aldehyde derivative of normetanephrine was produced in situ by monoamine oxidase, and two forms of aldehyde reductase were shown to metabolize the aldehyde to MOPEG. One form of the enzyme was found to have a low affinity for NADH and a higher affinity for NADPH as a cofactor, and was shown to be inhibited by pentobarbital and by high concentrations of 5-hydroxyindoleacetic acid. This enzyme form was localized primarily in the cytosol. The second aldehyde reductase had a high affinity for both NADH and NADPH, and was not inhibited to a great extent by either pentobarbital or 5-hydroxyindoleacetic acid. This second enzyme form was localized primarily in the mitochondrial fraction. The relative contribution of the two enzyme forms to MOPEG formation in homogenates was estimated, using the various inhibitors and cofactors.     

Subregional and Intracellular Distribution of NADP-Linked Malic Enzyme in Human Brain

High total activity (expressed as μmol/min/g of wet tissue or per milligram of DNA) and differential subregional distribution of NADP-linked malic enzyme was found in autopsy specimens of human brain. Striatum showed the highest activity of malic enzyme, which was two to five-fold higher than that in other human organs tested. High activity was also found in frontal cortex, while the lowest activity of the enzyme in the central nervous system was found in cerebellum, substantia alba, and corpus callosum. In striatum, frontal cortex, pens, and cerebellum more than 80% of total malic enzyme activity was localized in the mitochondrial fraction, while in substantia alba and corpus callosum approximately 60% of the enzyme activity was present in the mitochondrial fraction. Relatively high specific activity of malic enzyme was found in a crude mitochondrial fraction isolated from various regions of human brain. The highest specific activity was found in the mitochondria isolated from striatum (more than 100 nmol/min/mg of mitochondrial protein); the lowest, but still high (approximately 32 nmol/min/mg of mitochondrial protein) was present in corpus callosum. These data and the different ratios of citrate synthase to mitochondrial malic enzyme activities found in different regions of brain suggest that human brain mitochondria, like the mitochondria isolated from other mammalian brains, are extremely heterogenous. A possible role of mitochondrial malic enzyme in human brain metabolism is discussed.     

Uridine phosphorylase activity of isolated plasma membranes of rat liver.

Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5'-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5'-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible. The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.     

Subcellular distribution of carbonic anhydrase and Na+,K+-ATPase in the brain of the hyt/hyt hypothyroid mice

Activities of carbonic anhydrase and Na⁺,K⁺-ATPase in tissue homogenates and in subcellular fractions from different brain regions were studied in inherited primary hypothyroid (hyt/hyt) mice. The body weight, the weight of different brain regions, and the plasma thyroxine and triiodothyronine levels of hyt/hyt mice were significantly lower than those of the age-matched hyt/+ controls. In tissue homogenates of cerebral cortex, brain stem and cerebellum of hypothyroid mice, the activity of carbonic anhydrase (units/mg protein) was 59.2, 57.6, and 43.2%, and the activity of Na⁺,K⁺-ATPase (nmol Pi/mg protein/min) was 73.7, 74.4 and 68.7%, respectively, of that in corresponding regions of euthyroid littermates. The decrease in enzyme activity in tissue homogenates was also reflected in different subcellular fractions. In cerebral cortex and brain stem, carbonic anhydrase activity in cytosol, myelin and mitochondrial fractions of hypothyroid mice was about 45–50% of that in euthyroid mice, while in cerebellum the carbonic anhydrase activity in these subcellular fractions of hyt/hyt mice was only 33–38% of that in hyt/+ controls. Na⁺,K⁺-ATPase activity in myelin fraction of different brain regions of hyt/hyt mice was about 34–42% of that in hyt/+ mice, while in mitochondria, synaptosome and microsome fractions were about 44–52, 46–53, and 66–68%, respectively of controls. These data indicate that the activity of both carbonic anhydrase and Na⁺,K⁺-ATPase was affected more in the myelin than other subcellular fractions and more in the cerebellum than cerebral cortex and brain stem by deficiency of thyroid hormones. A reduction in the activity of transport enzymes in brain tissues as a result of thyroid hormone deficiency during the critical period of development may underlie permanent nervous disorders in primary hypothyroidism.     

4-Aminobutyraldehyde Dehydrogenase Activity in Rat Brain

Abstract: An enzyme with NAD⁺-dependent 4-aminobutyraldehyde dehydrogenase activity was purified about 360-fold from rat brain extract. AMP-Sepharose chromatography was effective in separating the enzyme from other NAD⁺-dependent aldehyde dehydrogenases included in the extract. The K _ms for the substrates NAD⁺ and 4-aminobutyraldehyde were 4.8 × 10⁻⁴ and 8.3 × 10⁻⁵ M , respectively. The pH optimum for the enzyme was about 8.0. The ratio of activities toward 4-aminobutyraldehyde, propionaldehyde, succinate semialdehyde, and benzaldehyde was 1.00:0.17:0.24:0.09:0.03 when the activity toward 4-aminobutyraldehyde was set equal to 1.00. The enzyme activity in subcellular fractions of rat brain was localized in cytosol.     

Agmatinase Activity in Rat Brain: A Metabolic Pathway for the Degradation of Agmatine

Abstract: Agmatinase, the enzyme that hydrolyzes agmatine to form putrescine and urea in lower organisms, was found in rat brain. Agmatinase activity was maximal at pH 8–8.5 and had an apparent K _m of 5.3 ± 0.99 m M and a V _max of 530 ± 116 nmol/mg of protein/h. After subcellular fractionation, most of the enzyme activity was localized in the mitochondrial matrix (333 ± 5 nmol/mg of protein/h), where it was enriched compared with the whole-brain homogenate (7.6–11.8 nmol/mg of protein/h). Within the CNS, the highest activity was found in hypothalamus, a region rich in imidazoline receptors, and the lowest in striatum and cortex. It is interesting that other agmatine-related molecules such as arginine decarboxylase, which synthesizes agmatine, and I₂ imidazoline receptors, for which agmatine is an endogenous ligand, are also located in mitochondria. The results show the existence of rat brain agmatinase, mainly located in mitochondria, indicating possible degradation of agmatine by hydrolysis at its sites of action.     

Intraparticulate localization and some properties of a clofibrate-induced peroxisomal aldehyde dehydrogenase from rat liver

A study was made of the effect of chronic administration of the hypolipidemic drug clofibrate on the activity and intracellular localization of rat liver aldehyde dehydrogenase. The enzyme was assayed using several aliphatic and aromatic aldehydes. Clofibrate treatment caused a 1.5 to 2.3-fold increase in the liver specific aldehyde dehydrogenase activity. The induced enzyme has a high Km for acetaldehyde and was found to be located in peroxisomes and microsomes. Clofibrate did not alter the enzyme activity in the cytoplasmic fraction. The total peroxisomal aldehyde dehydrogenase activity increased 3 to 4-fold under the action of clofibrate. Disruption of the purified peroxisomes by the hypotonic treatment or in the alkaline conditions resulted in the release of catalase from the broken organelles, while aldehyde dehydrogenase as well as nucleoid-bound urate oxidase and the peroxisomal membrane marker NADH:cytochrome c reductase remained in the peroxisomal 'ghosts'. At the same time, treatment by Triton X-100 led to solubilization of the membrane-bound NADH:cytochrome c reductase and aldehyde dehydrogenase from intact peroxisomes and their 'ghosts'. These results indicate that aldehyde dehydrogenase is located in the peroxisomal membrane. The peroxisomal aldehyde dehydrogenase is active with different aliphatic and aromatic aldehydes, except for formaldehyde and glyceraldehyde. The enzyme Km values lie in the millimolar range for acetaldehyde, propionaldehyde, benzaldehyde and phenylacetaldehyde and in the micromolar range for nonanal. Both NAD and NADP serve as coenzymes for the enzyme. Aldehyde dehydrogenase was inhibited by disulfiram, N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic)acid. According to its basic kinetic properties peroxisomal aldehyde dehydrogenase seems to be similar to a clofibrate-induced microsomal enzyme. The functional role of both enzymes in the liver cells is discussed.     

Metabolism of the lipid peroxidation product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic fractions.

The metabolism of the lipid peroxidation product 4-hydroxynonenal and of several other related aldehydes by isolated hepatocytes and rat liver subcellular fractions has been investigated. Hepatocytes rapidly metabolize 4-hydroxynonenal in an oxygen-independent process with a maximum rate (depending on cell preparation) ranging from 130 to 230 nmol/min per 10(6) cells (average 193 +/- 50). The aldehyde is also rapidly utilized by whole rat liver homogenate and the cytosolic fraction (140 000 g supernatant) supplemented with NADH, whereas purified nuclei, mitochondria and microsomes supplemented with NADH show no noteworthy consumption of the aldehyde. In cytosol, the NADH-mediated metabolism of the aldehyde exhibits a 1:1 stoichiometry, i.e. 1 mol of NADH oxidized/mol of hydroxynonenal consumed, and the apparent Km value for the aldehyde is 0.1 mM. Addition of pyrazole (10 mM) or heat inactivation of the cytosol completely abolishes aldehyde metabolism. The various findings strongly suggest that hepatocytes and rat liver cytosol respectively convert 4-hydroxynonenal enzymically is the corresponding alcohol, non-2-ene-1,4-diol, according to the equation: CH3-[CH2]4-CH(OH)-CH = CH-CHO + NADH + H+----CH3-[CH2]4-CH(OH)-CH = CH-CH2OH + NAD+. The alcohol non-2-ene-1,4-diol has not yet been isolated from incubations with hepatocytes and liver cytosolic fractions, but was isolated in pure form from an incubation mixture containing 4-hydroxynonenal, isolated liver alcohol dehydrogenase and NADH and its chemical structure was confirmed by mass spectroscopy. Compared with liver, all other tissues possess only little ability to metabolize 4-hydroxynonenal, ranging from 0% (fat pads) to a maximal 10% (kidney) of the activity present in liver. The structure of the aldehyde has a strong influence on the rate and extent of its enzymic NADH-dependent reduction to the alcohol. The saturated analogue nonanal is a poor substrate and only a small proportion of it is converted to the alcohol. Similarly, nonenal is much less readily utilized as compared with 4-hydroxynonenal. The effective conversion of the cytotoxic 4-hydroxynonenal and other reactive aldehydes to alcohols, which are probably less toxic, could play a role in the general defence system of the liver against toxic products arising from radical-induced lipid peroxidation.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

Biosynthesis of N-acylethanolamine phospholipids by dog brain preparations

Abstract: Dog brain homogenates and subcellular preparations incubated in the presence of Ca²⁺ produced a new phospholipid that was isolated and identified by its infrared spectrum and by chemical degradation as a mixture of 1, 2-diacyl, alkenylacyl, and alkylacyl sn -glycero-3-phospho ( N -acyl)ethanolamines, 50, 45, and 5%, respectively. The N -acyl groups consisted almost exclusively of 16:0, 18:0, and 18:1 fatty acids. Formation of N -acylethanolamine phospholipids from endogenous substrates was linear for about 90 min at approximately 4.5 nmol/h/mg protein and exhibited a pH optimum of 10. Biosynthetic activity was associated with particulate fractions, primarily microsomes, synaptosomes, and mitochondria, but not with myelin. In each case, small amounts (∼0.5 nmol/h/mg protein) of long-chain N -acylethanolamines were also produced. Incubation of dog brain microsomes with 1,2-di[1'-¹⁴C]palmitoyl glycero-phosphocholine yielded N -acylethanolamine phospholipids labeled at both N -acyl (55%) and O -acyl (45%) moieties. It appears that dog brain organelles may contain a phosphatidylethanolamine N -acyl transferase (transacylase) analogous to that recently demonstrated in the myocardial tissue.     

Phosphatidylinositol kinase,phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [γ-³²P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150–200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4–11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40–70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP₂ in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.     

Purification and characterization of an NADH-linked aldehyde reductase from bovine brain

Abstract— The presence of a nonspecific NADH-linked aldehyde reductase was demonstrated in various regions of bovine brain in vitro. With m-nitrobenzaldehyde as substrate, the rate of NADH oxidation was approximately 4 nmol.min^-1.(mg of protein)^-1 in the cerebellum, pons and medulla; but somewhat lower rates [2–3 nmol.min^-1.(mg of protein)-^l] were obtained in the other areas of the brain examined. The enzyme was localized primarily in the soluble, supernatant fraction of rat brain homogenates. The enzyme from the supernatant fluid fraction of bovine brain was purified approximately 350-fold by ammonium sulphate fractionation and chromatography on calcium phosphate-gel, DEAE-cellulose and Sephadex G200 columns. The partially purified enzyme catalysed the reduction of a number of aldehydes, including substituted benzaldehydes and aliphatic aldehydes of intermediate chain lengths. Short chain aliphatic aldehydes, such as acetaldehyde, were not reduced by the enzyme and butyraldehyde was a poor substrate. With m-nitrobenzaldehyde as substrate, NADH was oxidized at an approximately 10-fold faster rate than NADPH. The pH optimum for the enzyme was 6.75 for aldehyde reduction, whereas the rate of oxidation of m-nitrobenzylalcohol was optimal at pH 10.0 with NAD as the co-substrate. K_m and K₃ values ranged from 10 μM to 10 mM for various aldehydes and from 10 to 30 μM for the cofactors. Oxidation of NADH by the partially purified enzyme was not inhibited by 10m pyrazole or by 1 mM phenobarbital. However, the enzyme activity was inhibited by approximately 60 percent by 1 mM chlorpromazine or by 5 mM 1,10-orthophenanthroline. Our data demonstrate that the enzyme is not only separable from the NADPH-linked aldehyde reductase described previously by TABAKOFF and ERWIN, but also is quite different in substrate specificity and inhibitor sensitivity from the ‘classical’, pyrazole-sensitive, NAD- linked alcohol dehydrogenase (EC 1.1.1.1).     

Nicotinamide cofactors (NAD and NADP) in glyoxysomes,mitochondria, and plastids isolated from castor bean endosperm

Glyoxysomes, mitochondria, and plastids were separated from the cytosol of germinating castor bean endosperm by sucrose gradient centrifugation in a vertical rotor (25 min, 50,000g_av). The amounts of nicotinamide cofactors, NAD(H) and NADP(H), retained in the isolated organelle fractions were measured by enzyme cycling techniques. The NAD(H) was equally distributed between the cytosol and the mitochondria with a small amount in the glyoxysomes. The mitochondria retained 4 pmol of NAD(H)/ μg protein, about seven times as much as the glyoxysomes. Most of the NADP(H) was in the cytosol. However, the glyoxysomes and plastids retained significant amounts, both having 0.3 pmol NADP(H)/μg protein, twice that in the mitochondria. The subcellular distribution of NADP(H) was compared to the location of dehydrogenases capable of using this cofactor. The cytosol and plastids contained 6-phosphogluconate dehydrogenase. NADP isocitrate dehydrogenase was found in the glyoxysomes, in mitochondria, and in an unidentified subcellular fraction obtained at 1.16 g/ml in the density gradients. Knowledge of the quantities of NADP(H) and NAD(H) retained in the isolated organelles should make it possible to investigate their reduction and reoxidation in intact organelles.     

THE CONTROL OF PYRUVATE DEHYDROGENASE IN ISOLATED BRAIN MITOCHONDRIA

Abstract— The activity and control of the pyruvate dehydrogenase complex in isolated rat brain mitochondria has been studied. The activity of this complex in mitochondria as isolated from normal fed rats was 78 ± 10nmol.min⁻¹ mg mitochondrial protein⁻¹ (n = 18) which represented 70% of the total pyruvate dehydrogenase activity. The pyruvate dehydrogenase in isolated brain mitochondria could be inactivated by incubation in the presence of ATP, oligomycin and NaF. The rate of inactivation was dependent upon the added ATP concentration but inactivation below approx 30% of the total pyruvate dehydrogenase activity could not be achieved. The inactivation of pyruvate dehydrogenase in brain mitochondria was inhibited by pre-incubation with pyruvate. Reactivation of inactivated pyruvate dehydrogenase in rat brain mitochondria was incomplete in the incubation medium unless 10mM-Mg²⁺+ 1 mM-Ca²⁺ were added; NaF, however, prevented any reactivation (Fig. 4). It is concluded that the pyruvate dehydrogenase complex in rat brain mitochondria is controlled in a manner similar to that in other tissues, and that pyruvate protection of pyruvate dehydrogenase activity may be important in maintaining brain energy metabolism.     

The effect of ethanol ingestion on the aldehyde dehydrogenases of rat liver

The effect of ethanol ingestion on aldehyde dehydrogenase activity in the subcellular fractions of livers from 14 pair-fed male Sprague-Dawley rats was tested. Enzymatic assays were performed at two different concentrations of propionaldehyde (0.068 and 13.6 mM) sufficient to saturate enzymes with high and low affinities for propionaldehyde, respectively. The effect of alcohol ingestion varied depending on the subcellular fraction tested and the propionaldehyde concentration used in the assay. There was a 60% increase in the activity of aldehyde dehydrogenase with high affinity for propionaldehyde in the mitochondrial membranes. Conversely there was a 50% decrease in the activity of aldehyde dehydrogenases with high affinity for propionaldehyde in the microsomal fraction. There was also a 58% decrease in the activity of enzymes from the mitochondrial matrix with low affinity for propionaldehyde. The results suggest that differences in the assay systems employed may account for the conflicting results obtained by previous investigators of the effect of ethanol feeding.     

Comparative subcellular distribution of benzaldehyde and acetaldehyde dehydrogenase activities in two hepatoma cell lines and in normal hepatocytes.

The NAD- and NADP-dependent aldehyde dehydrogenase (ALDH) activities were evaluated in two rat hepatoma cell lines, namely the well-differentiated MH1C1 line and the less differentiated HTC line. Each activity was determined in parallel in isolated rat hepatocytes, for comparison. The aliphatic aldehyde acetaldehyde (ACA) and the aromatic aldehyde benzaldehyde (BA) were used as substrates. With the first substrate the ALDH activities found in the crude cytoplasmic extracts were lower in hepatoma cells than in normal hepatocytes, especially when measured with NADP as coenzyme (ACA/NADP). Otherwise, with benzaldehyde as substrate the NAD-dependent enzyme activity (BA/NAD) was increased about 9-fold in HTC cells over hepatocytes and decreased in MH1C1 cells, while the NADP-dependent (BA/NADP) activity was increased 38- and 2.5-fold in HTC and MH1C1 cell lines, respectively. Studies on the subcellular distribution of these enzyme activities showed that the activity measured with acetaldehyde and NAD (ACA/NAD) was almost equally distributed between the cytosol and the subcellular particles in the three cell populations, but the ACA/NADP activity was shifted towards the cytosolic compartment in hepatomas, especially in HTC cells. The BA/NAD and BA/NADP ALDH activities found in the organelles of hepatoma cells were markedly reduced in comparison with hepatocytes, in favour of the cytosol. The most striking difference between the normal and the transformed cells was the 94-fold increase over hepatocytes of the BA/NADP activity, found in the cytosolic fractions of HTC cells. MH1C1 cells showed a less pronounced (7.5-fold) enhancement of this tumour-associated specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of ethanol ingestion on the aldehyde dehydrogenases of rat liver.

The effect of ethanol ingestion on aldehyde dehydrogenase activity in the subcellular fractions of livers from 14 pair-fed male Sprague-Dawley rats was tested. Enzymatic assays were performed at two different concentrations of propionaldehyde (0.068 and 13.6 mM) sufficient to saturate enzymes with high and low affinities for propionaldehyde, respectively. The effect of alcohol ingestion varied depending on the subcellular fraction tested and the propionaldehyde concentration used in the assay. There was a 60% increase in the activity of aldehyde dehydrogenase with high affinity for propionaldehyde in the mitochondrial membranes. Conversely there was a 50% decrease in the activity of aldehyde dehydrogenases with high affinity for propionaldehyde in the microsomal fraction. There was also a 58% decrease in the activity of enzymes from the mitochondrial matrix with low affinity for propionaldehyde. The results suggest that differences in the assay systems employed may account for the conflicting results obtained by previous investigators of the effect of ethanol feeding.     

A mitochondrial NADP+-dependent reductase related to the 4-aminobutyrate shunt. Purification, characterization, and mechanism

Succinic semialdehyde reductase, a NADP+-dependent enzyme, was purified from whole pig brain homogenates. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Succinic semialdehyde reductase (Mr 110,000) catalyzes the reduction of succinic semialdehyde to 4-hydroxybutyrate. The equilibrium constant of the reaction is Keq = 5.8 X 10(7) M-1 at pH 7 and 25 degrees C. The inhibition kinetic patterns obtained when 4-hydroxybutyrate or substrate analogs are used as inhibitors of the reaction catalyzed by the reductase are consistent with an ordered sequential mechanism, in which the coenzyme NADPH adds to the enzyme before the aldehyde substrate. A specific aldehyde reductase was also purified to homogeneity from brain mitochondria preparations. Its catalytic properties are identical to those of the enzyme isolated from whole brain homogenates. It is postulated that two enzymes, i.e. a NAD+-dependent dehydrogenase and a NADP+-dependent reductase, participate in the metabolism of succinic semialdehyde in the mitochondria matrix.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.