Formation of 2-methyl-2,4-thiazolidinedicarboxylic acid from L-cysteine in rat tissues

The adduct formed non-enzymatically from L-cysteine and pyruvate: 2-methyl-2,4-thiazolidinecarboxylic acid (CP) was isolated, and identified by the electron impact mass spectroscopy. It was found that CP is formed (by cysteine transformation) and is metabolized in rat tissues. Formation of CP from cysteine or cystine was catalysed by partially purified rat liver gamma-cystathionase.     

Determination of rhodanese activity by tetrazolium reduction

A colorimetric method for the assay of rhodanese activity based on the continuous determination of the sulfite product is described. 5-Ethylphenazinium ethyl sulfate is used as the intermediate electron carrier between sulfite and nitroblue tetrazolium to produce the colored reduced species. The present method is more sensitive than the usual procedure based on the colorimetric determination of thiocyanate. Furthermore, the color developed by nitroblue tetrazolium reduction affords a straightforward means to locate rhodanese activity in polyacrylamide gels.     

Pyridoxal 5'-phosphate dependent enzymes in the nematode Nippostrongylus brasiliensis.

Eight classes of pyridoxal 5'-phosphate dependent enzymes have been investigated in Nippostrongylus brasiliensis in parallel with rat tissues. The range of decarboxylases detected in N. brasiliensis was limited in comparison with rat tissues. N. brasiliensis possessed a highly active L-serine hydroxymethyltransferase, but in contrast with rat liver, 5-aminolevulinic acid synthetase was absent. Similar levels of L-serine and L-threonine dehydratase activities were detected in N. brasiliensis and rat liver, and both organisms lacked L-alanine racemase, L-tryptophan synthetase and L-methionine gamma-lyase. The demonstration of cystathionine beta-synthase and gamma-cystathionase in N. brasiliensis suggests the presence of a functional trans-sulphuration sequence. The substrate specificities of the nematode cystathionine beta-synthase and gamma-cystathionase varied significantly from those of the corresponding mammalian enzymes. Particularly striking was the ability of N. brasiliensis cystathionine beta-synthase to catalyse the non-mammalian 'activated L-serine sulphydrase' reaction (L-cysteine + R-SH----cysteine thioether + H2S). N. brasiliensis and rat liver exhibited comparable abilities to transaminate amino acids via the 2-oxoglutarate: glutamate system.     

Purification and characterization of selenocysteine beta-lyase from Citrobacter freundii.

The purification and characterization of bacterial selenocysteine beta-lyase, an enzyme which specifically catalyzes the cleavage of L-selenocysteine to L-alanine and Se0, are presented. The enzyme, purified to near homogeneity from Citrobacter freundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 mol of pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate (Km, 0.95 mM). L-Cysteine is a competitive inhibitor of the enzyme (Ki, 0.65 mM). The enzyme also catalyzes the alpha, beta elimination of beta-chloro-L-alanine to form NH3, pyruvate, and Cl- and is irreversibly inactivated during the reaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterial enzyme are fairly different from those of the pig liver enzyme (Esaki et al., J. Biol. Chem. 257:4386-4391, 1982). However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by the substrate or a mechanism-based inactivator, beta-chloro-L-alanine, are very similar.     

Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.     

Immunochemical studies with soluble and mitochondrial pyruvate carboxylase activities from rat tissues

1. Pyruvate carboxylase (EC 6.4.1.1), purified from rat liver mitochondria to a specific activity of 14 units/mg, was used for the preparation of antibodies in rabbits. 2. Tissue distribution studies showed that pyruvate carboxylase was present in all rat tissues that were tested, with considerable activities both in gluconeogenic tissues such as liver and kidney and in tissues with high rates of lipogenesis such as white adipose tissue, brown adipose tissue, adrenal gland and lactating mammary gland. 3. Immunochemical titration experiments with the specific antibodies showed no differences between the inactivation of pyruvate carboxylase from mitochondrial or soluble fractions of liver, kidney, mammary gland, brown adipose tissue or white adipose tissue. 4. The antibodies were relatively less effective in reactions against pyruvate carboxylase from sheep liver than against the enzyme from rat tissues. 5. Pyruvate carboxylase antibodies did not inactivate either propionyl-CoA carboxylase or acetyl-CoA carboxylase from rat liver. 6. It is concluded that pyruvate carboxylase in lipogenic tissues is similar antigenically to the enzyme in gluconeogenic tissues and that the soluble activities of pyruvate carboxylase detected in many rat tissues do not represent discrete enzymes but are the result of mitochondrial damage during tissue homogenization.     

Micromethods for the study of hexokinase in human erythrocytes

A direct radioassay for the erythrocyte enzyme using U14C-glucose as substrate has been developed. With respect to the indirect spectrophotometric assay this method allows for the determination of true hexokinase activity. The assay proposed is sensitive, rapid and well suited for the determination of hexokinase activity in the erythrocyte lysate where the enzyme level is particularly low.     

Cooperative interaction of pyruvate dehydrogenase from pigeon breast muscle]

Cooperative interaction of pyruvate with the pyruvate dehydrogenase (PD) complex from pigeon breast muscle was shown. The sigmoidal dependence of the reaction rate on pyruvate concentration was observed for the PD complex. The Hill coefficient is equal to 1,5; no inhibition by the substrate (up to 2.2.10(-3) M) was found. The kinetic behaviour of the isolated pyruvate dehydrogenase component (PDH) analyzed under similar conditions, is more complex; this may be probably due to the presence of oligomeric forms with different molecular weights and specific activities. The competitive inhibitor of the PD complex--an amide of pyruvic acid (PA) (Ki=6.3-10(-6) M) activates the enzyme at low concentrations (less than 2,10(-6) M). When PA is present, the dependence of the reaction rate on pyruvate concentration gives a usual hyperbolic curve, v of [S]o. It is concluded that pyruvate may have a regulatory effect on the activity of muscle PD complex.     

A highly sensitive high-performance liquid chromatography-- fluorometric method for the assay of peptidylarginine deiminase activity

The activity of peptidylarginine deiminase (PAD) has generally been assayed by a colorimetric method using N-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-arginine (Bz-L-Arg) as the substrates. The widespread occurrence of citrulline and urea in tissues makes use of this method difficult, especially for small samples. We developed a highly sensitive high-performance liquid chromatography method with N-dansyl-glycyl-L-arginine as the substrate. This method was sensitive enough to determine previously undetectable activity of PAD in HL-60 cells. Two types of PAD (HL-60 cell and brain PAD) could be distinguished by differential competition, using either BAEE or Bz-L-Arg as a preferential substrate in the assay. These data indicate that the present method is applicable to many tissues.     

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.     

Development of gluconeogenesis in neonatal rat liver. Effect of premature delivery

1. An assay method for the determination of phosphopyruvate carboxylase activity is described in which improved sensitivity is obtained by separation of the enzyme from interfering pyruvate kinase by zone sedimentation. 2. The molecular weight of rat liver phosphopyruvate carboxylase determined by zone sedimentation is about 68000. 3. Premature delivery of rat foetuses by uterine section results in the rapid appearance of phosphopyruvate carboxylase, but hexose diphosphatase and pyruvate carboxylase, already present in the foetal rat liver, are not significantly affected, and glucose 6-phosphatase activity is only slightly affected. 4. The rate of incorporation of [¹⁴C]pyruvate into glucose by liver slices is also greatly increased by premature delivery and there is a highly significant linear correlation between this process and the phosphopyruvate carboxylase activity.     

Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis.

The pyruvate dehydrogenase complex of Lactococcus lactis subsp. lactis bv. diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate. The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively. The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa). The L. lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruvate. Thiamine pyrophosphate (Km = 3.2 microM) and divalent cations are essential for activity. The native enzyme measures 172 kDa and consists of 62-kDa monomers. The role of both enzymes in product formation is discussed in view of NADH inhibition and competition for pyruvate.     

Highly sensitive high-performance liquid chromatography-fluorimetric assay method for carboxypeptidase H activity

A rapid and sensitive high-performance liquid chromatographic (HPLC)-fluorimetric assay method has been developed for the determination of carboxypeptidase H activity based on the measurement of N-(5-dimethyl-aminonaphthalene-1-sulfonyl)glycine (dansyl-Gly) formed enzymatically from dansyl-Gly-L-Lys or dansyl-Gly-L-Arg. Dansyl-Gly is eluted faster than the substrates with an N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes) buffer at pH 7.0 containing methanol, but eluted slower with an acidic buffer at pH 4.6. The new HPLC method separates the product and substrate in less than 5 min using an elution buffer at pH 7.0 containing 60% methanol. Using this method carboxypeptidase H activity has been detected in rat sciatic nerves. This HPLC method facilitates the assay of carboxypeptidase H activity in the enzyme samples from various tissues.     

Comparison of Isocitrate Dehydrogenase Activity, Pyruvate Kinase Activity, and Polyphenol Content in Physiologically Different Pine Callus Tissue

During the growth of callus tissue of slash pine (Pinus elliottil Engelm.) several physiologically different types of tissue can be observed, often within the same culture. Different tissues were selected, based on color appearance, and used to determine isocitrate dehydrogenase and pyruvate kinase activity, and total polyphenol content. Isocitrate dehydrogenase and pyruvate kinase activity in yellow tissue was 3- to 5-fold greater than in brown tissue, whereas the polyphenol content in yellow tissue was approximately 5-fold less than in brown tissue. Dark brown callus, which also contained large amounts of polyphenols, did not have detectable enzyme activity. The differences in optimal concentrations of substrate and cofactors for the isocitrate dehydrogenase and pyruvate kinase reactions in yellow and brown tissues were very minor and therefore cannot account for the 3- to 5-fold difference in enzyme activity between these tissues. Also, the addition of brown or dark-brown tissue extract to the yellow tissue extract did not inhibit isocitrate dehydrogenase or pyruvate kinase activity in the yellow tissue extract.     

Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium

Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue protectant and Tetranitro BT as final electron acceptor. Control media lacked either substrate or substrate and coenzyme. Controls were also performed by adding malonate (a competitive inhibitor of succinate dehydrogenase), pyruvate (a non-competitive inhibitor of lactate dehydrogenase), oxalate (a competitive inhibitor of lactate dehydrogenase) or N-ethylmaleimide (a blocker of SH groups). A specific malonate-sensitive linear test minus control response for succinate dehydrogenase activity was obtained in liver (1.6 mumol H2cm-3 min-1) and tracheal epithelium (0.8 mumol H2cm-3 min-1) but not in heart muscle. All variations in the incubation conditions tested did not result in a linear test minus control response in the latter tissue. Because the reaction was sensitive to malonate, it was concluded that the initial reaction rate was the specific rate of succinate dehydrogenase activity in heart muscle (9.1 mumol H2 cm-3 min-1). Test minus control reactions for lactate dehydrogenase activity were distinctly non-linear for all tissues tested. This appeared to be due to product inhibition by pyruvate generated during the reaction and therefore it was concluded that the appropriate control reaction was the test reaction in the presence of 20 mM pyruvate. The initial rate of the test minus this control was the true rate of lactate dehydrogenase activity. The lactate dehydrogenase activity thus found in liver parenchyma was 5.0 mumol of H2 generated per cm3 liver tissue per min.     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

Growth-rate-related and hydroxysterol-induced changes in membrane fluidity of cultured hepatoma cells: correlation with 3-hydroxy-3-methyl glutaryl CoA reductase activity

Creatine kinase (CK) activity in plasma obtained non-invasively from adult healthy, Sprague-Dawley, male rats was found to be 528 +/- 270 U/L (N = 17), a value which was 7 times that obtained in human specimens. Agarose gel electrophoresis revealed that the only detectable CK isoenzyme present was CK-BB, in contrast to the human serum isoenzyme which was CK-MM. Furthermore, it was found that the rat CK-BB could be detected using an RIA technique designed to quantitate human CK-BB occasionally present in blood after brain injury (rat CK-BB = 84.5 +/- 55.2 micrograms/L, N = 17, human CK-BB: Not detectable). It was thus possible to calculate the CK-BB specific activity (SA) in rat plasma using total CK assay and RIA (rat CK-BB SA = 6.25 +/- 3.87 U/micrograms, N = 17). When six rats (156 +/- 23 g) were treated with lead acetate in the drinking water (26 mM) for 3 weeks, the CK-BB SA rose to 18 +/- 5.8 U/micrograms (P less than .02). At this point the electrophoresis pattern of the CK-BB showed a transient change from a single band to a doublet. The dose was then increased to 52 mM for 6 weeks, during which time the CK-BB SA declined steadily to 1.6 +/- 0.6, a level significantly less than that of the untreated animals (p less than .02). The results suggest that chronic lead treatment evokes a biphasic response in CK-BB SA with the initial release of enzyme of high SA from tissues. Further treatment apparently results in an inactivation of the enzyme within lead sensitive tissues.     

The activities and intracellular distribution of nicotinamide-adenine dinucleotide phosphate-malate dehydrogenase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in rat, guinea-pig and rabbit tissues.

1. Measurements are presented of the activity and intracellular distribution of phosphoenolypruvate carboxykinase, pyruvate carboxylase and NADP-malate dehydrogenase in rat, guinea-pig and rabbit liver and kidney cortex, together with previously obtained measurements of these enzymes in adipose tissue. 2. In all three tissues pyruvate carboxylase activity was greatest in the rat and lowest in the rabbit. 3. Guinea pig and rabbit were very similar to each other with respect to the extramitochondrial-mitochondrial distribution of phosphoenolpyruvate carboxykinase in all three tissues. 4. NADP-malate dehydrogenase was present in all three tissues in the rat, present in kidney cortex and adipose tissue in the guinea pig and absent from all tissues examines in the rabbit.     

Kinetic measurement by LC/MS of gamma-glutamylcysteine ligase activity

gamma-Glutamylcysteine ligase (GCL) combines cysteine and glutamate through its gamma carboxyl moiety as the first step for glutathione (GSH) synthesis and is considered to be the rate-limiting enzyme in this pathway. The enzyme is a heterodimer, with a heavy catalytic and a light regulatory subunit, which plays a critical role in the anti-oxidant response. Besides the original method of Seelig designed for the measurement of a purified enzyme, few endpoint methods, often unrefined, are available for measuring it in complex biological samples. We describe a new, fast and reliable kinetic LC/MS method which enabled us to optimize its detection. l-2-Aminobutyrate is used instead of cysteine (to avoid glutathione synthetase interference) as triggering substrate with saturating concentrations of glutamate and ATP; the gamma glutamylaminobutyrate formed is measured at m/z=233 at regular time intervals. Reaction rate is maximum because ATP is held constant by enzymatic recycling of ADP by pyruvate kinase and phosphoenolpyruvate. The repeatability of the method is good, with CV% of 6.5 and 4% for catalytic activities at, respectively 0.9 and 34 U/l. The affinities of rat and human enzymes for glutamate and aminobutyrate are in good agreement with previous published data. However, unlike the rat enzyme, human GCL is not sensitive to reduced glutathione and displays a more basic optimum pH.     

Regulation of citric acid cycle by calcium

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.