ASSAY AND PROPERTIES OF 4-AMINOBUTYRIC-2-OXOGLUTARIC ACID TRANSAMINASE AND SUCCINIC SEMIALDEHYDE DEHYDROGENASE IN RAT BRAIN TISSUE

Abstract— The activity of 4-aminobutyric-2-oxoglutaric acid transaminase (GABA transaminase) and succinic semialdehyde dehydrogenase was determined in total rat brain homogenate. GABA transaminase activity was measured using a coupled enzyme method which utilizes endogenous succinic semialdehyde dehydrogenase to convert the formed succinic semialdehyde into succinate. The concurrently produced NADH was used as an estimate of GABA transaminase activity. This method could be used since it was shown that the dehydrogenase was about twice as active as the transaminase and because no significant accumulation of the intermediate succinic semialdehyde could be detected. GABA transaminase was inhibited by high ionic strength. In contrast NaCl decreased the apparent K _m and increased V _max for succinic semialdehyde dehydrogenase at high but not al low tissue concentrations. Increasing tissue concentration also resulted in a decrease of the apparent K _m, but did not change the V_max of succinic semialdehyde dehydrogenase and it is suggested that this enzyme can exist in two distinct states of aggregation, one with a high and one with a low affinity for succinic semialdehyde. The high affinity form of the enzyme is thought to prevent succinic semialdehyde from accumulation in the GABA transaminase assay. It is concluded that within certain limits the coupled enzyme method described here can be used for the assay of GABA transaminase activity.     

PREPARATION OF SUCCINIC SEMIALDEHYDE: ITS COLORIMETRIC ESTIMATION AS AN ASSAY FOR GABA AMINOTRANSFERASE

Abstract— A simple method is described for the preparation of succinic semialdehyde by the oxidation of 4-hydroxybutyric acid with dimethyl sulphoxide in the presence of phosphorus pentoxide. The colorimetric estimation of succinic semialdehyde by condensation with 3-methyl-2-benzothiazolinone-2-hydrazone is used as an assay for GABA aminotransferase activity in mouse brain. The values obtained for the specific activity of this enzyme in mouse brain using the colorimetric method agree with those obtained by the more conventional isotopic method.     

GABA IN THE OLFACTORY BULB AND OLFACTORY NUCLEUS OF THE RAT: THE DISTRIBUTION OF GAMMA-AMINOBUTYRIC ACID, GLUTAMIC ACID DECARBOXYLASE, GABA TRANSAMINASE AND SUCCINATE SEMIALDEHYDE DEHYDROGENASE

Abstract— The distribution of GABA and enzymes involved in its metabolism was investigated in the different regions of the olfactory bulb and olfactory nucleus. The highest levels of GABA in the olfactory bulb were found in the layers rich in nerve terminals (31 μmol/g dry wt.). A similar distribution was found in the olfactory nucleus although the overall level of GABA was only a quarter of that measured in the bulb. Glutamic acid decarboxylase (GAD) levels in the various layers of the olfactory nucleus were similar in distribution to those of GABA. However, the correlation between GAD and GABA did not hold for the olfactory bulb, particularly in the granule cell layer and the medulla. The activities of GAD and the levels of GABA are significantly higher in the bulb than in the nucleus but succinic acid scmialdehyde dehydrogenase and GABA aminotransaminase activities are almost identical in both regions.     

THE DISTRIBUTION OF HISTOCHEMICALLY DEMONSTRABLE SUCCINIC DEHYDROGENASE AND OF MITOCHONDRIA IN TONGUE AND SKELETAL MUSCLES

Various skeletal muscles show considerable variations in histochemically demonstrable succinic dehydrogenase activity. In muscles from the lower extremities, the back, and the abdominal wall, only some fibers reveal evidence of enzymatic staining. In the diaphragm and masseter more fibers react positively. In the tongue, however, all fibers show marked activity comparable to that found regularly in the heart. There is a close similarity in the distribution of histochemically demonstrable succinic dehydrogenase and stainable mitochondria in tissue sections.     

KINETIC AND MOLECULAR MODELING OF NUCLEOSIDE AND NUCLEOTIDE INHIBITION OF MALATE DEHYDROGENASE

ABSTRACT

We studied the inhibition of mitochondrial malate dehydrogenase (mMDH) by the nucleotides cAMP, AMP, ADP, ATP. The experimental kinetic studies showed that the nucleotides were competitive inhibitors and that cAMP was probably the most potent inhibitor. To explain these observations, we used molecular modeling to determine the location, orientation, and relative binding energy of the nucleotides to mMDH. The order of the calculated binding energies, from lowest (most favorable) to highest, was cAMP, AMP, ADP, and ATP, which corresponded somewhat to the order of the experimentally determined inhibition constants.     

FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE OF MITOCHONDRIA

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q₁₀, and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5° to 10°C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro—BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.     

PURIFICATION FROM HUMAN BRAIN AND SOME PROPERTIES OF TWO NADPH-LINKED ALDEHYDE REDUCTASES WHICH REDUCE SUCCINIC SEMIALDEHYDE TO 4-HYDROXYBUTYRATE

Abstract— Two NADPH-linked aldehyde reductases (alcohol:NADP⁺oxidoreductase, EC 1.1.1.2) capable of reducing succinic semialdehyde to the anaesthetic Chydroxybutyrate have been purified from human brain to electrophoretic homogeneity. The first of these enzymes, which is typical of its category, is not specific for succinic semialdehyde and can reduce some aromatic aldehydes at a high rate. It is a monomer of molecular weight about 45,000 and is strongly inhibited by various hypnotics and anticonvulsants. The second enzyme is, in contrast, fairly specific for succinic semialdehyde. It is a dimer of molecular weight about 90,000 and is not inhibited by the hypnotics and anticonvulsants which inhibit the first enzyme. It is thus different from previously described aldehyde reductases from human brain.     

EFFECTS OF VASOACTIVE INTESTINAL PEPTIDE ON ACTIVITIES OF SUCCINIC DEHYDROGENASE AND ALKALINE PHOSPHATASE IN RAT HEPATOMA CELLS

Using cytochemical method,microspectrophotometry and image analysis,effects of va-soactive intestinal peptide(VIP)on activities of succinic dehydrogenase(SDH)and alkalinephosphatase(ALP)in rat hepatoma cells were studied in vitro.The results showed that thehepatoma cell expressed potent positive reactions of SDH and ALP,the positive positionswere located at the cell membranes and/or cytoplasm.Having been treated with VIP,ALPdecreased obviously in activity(P<0. 01,compared with hepatoma cells untreated by VIP).The sites of ALP activty were chiefly located at the cell membranes,particularly at the cell-cell contacts.Cultured rat hepatoma cells had intensive SDH activity in their cytoplasm.Compared with untreated eclls,there was no marked difference in the intensity of SDH activ-ity in VIP-treated hepatoma cells(P>0.05).     

LOCALIZATION OF THE SUCCINIC DEHYDROGENASE SYSTEM IN ESCHERICHIA COLI USING COMBINED TECHNIQUES OF CYTOCHEMISTRY AND ELECTRON MICROSCOPY

The activity of the succinic dehydrogenase system was studied in Escherichia coli utilizing combined techniques of cytochemistry and electron microscopy. Organisms were incubated in a medium containing tetranitro-blue tetrazolium (TNBT) which served as an electron acceptor. Enzymatic activity, as evidenced by the deposition of aggregates of TNBT-formazan, was found associated with the site of the plasma membrane of the bacterium.     

耳蜗琥珀酸脱氢酶显示方法的改进

在采用耳蜗顶部灌流方法测定琥珀酸脱氢酶的活性时,本文提出了两点改进,实验动物为豚鼠:(1)孵育前用2%戊二醛磷酸盐缓冲液(pH7.4,4℃)将耳蜗预固定30min,再冰凉(—20——40℃)约3min,以便消除血管纹屏障,使该部位和柯蒂氏器官的琥珀酸脱氢酶活性能同时被显示出来。(2)在孵育液中加入细胞呼吸抑制剂(阿密妥),可使耳蜗背景色变淡,从而减少假阳性现象。通过对一些豚鼠的实验观察,所得结果基本一致。     

纤维二糖脱氢酶的纤维素降解中的作用研究

裂褶菌纤维二糖脱氢酶（ｃｅｌｌｏｂｉｏｓｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ,ＣＤＨ）可以提高纤维素酶对纤维素的降解。以纤维二糖为电子供体,ＣＤＨ作用于羧甲基纤维可降低其溶液的粘度,作用纤维素ＣＦ１１和磷酸膨胀纤维素,分别使其悬浊液的浊度提高７％和１４．４％。ＣＤＨ与纤维二糖水解酶或切纤维素酶在降解棉花纤维素时没有表现出协同作用。但若棉花事先在纤维二糖存在下用ＣＤＨ预处理,则变得易于被水解。     

抑制伏核内脑啡肽的降解使电针镇痛和吗啡镇痛得到加强

将“脑啡肽酶”抑制剂 Thiorphan 或氨肽酶抑制剂 Bestatin 经慢性埋植套管注入家兔一侧状核内,观察到明显的镇痛作用,在1—4μg 范围内呈现明确的剂量-效应关系。该作用可为伏核内注射纳洛酮或甲啡肽抗体所完全翻转,亮啡肽抗体则无效。表明伏核内注射 Thior-Phan 或 Bestatn 所产生的镇痛效应主要是通过甲啡肽而完成的。伏核内注射微量 Thiorphan 或 Bestatin 使电针镇痛的后效应明显加强,并能增强吗啡的镇痛作用。表明电针和吗啡的镇痛效果至少有一部分是通过在伏核内释放出脑啡肽(特别是甲啡肽)而实现的。     

纤维二糖脱氢酶在纤维素降解中的作用研究*

裂褶菌纤维二糖脱氢酶（ｃｅｌｌｏｂｉｏｓｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ, ＣＤＨ）可以提高纤维素酶对纤维素的降解。以纤维二糖为电子供体, ＣＤＨ作用于羧甲基纤维素可降低其溶液的粘度,作用于纤维素 ＣＦ１１和磷酸膨胀纤维素,分别使其悬浊液的浊度提高７％和１４．４％。ＣＤＨ与纤维二糖水解酶或内切纤维素酶在降解棉花纤维素时没有表现出协同作用。但若棉花事先在纤维二糖存在下用ＣＤＨ预处理,则变得易于被水解。     

STUDIES ON THE MECHANISM OF HYDROGEN TRANSPORT IN ANIMAL TISSUES : VI. INHIBITOR STUDIES WITH SUCCINIC DEHYDROGENASE

1. The mechanism of succinic dehydrogenase action was studied by means of inhibitors. 2. The enzyme is inhibited by a large number of diverse compounds whose only common denominator appears to be their ability to react with SH groups. These compounds include quinonoid structures, sulfhydryl reagents, sulfhydryl compounds, copper, zinc, selenite, and arsenite. 3. In contrast to the above inhibitors, the action of malonate does not appear to involve sulfhydryl groups and is explained on the basis of its affinity for the enzyme groups which react with the carboxyl groups of succinate. 4. The action of malonate and the sulfhydryl reactants is mutually exclusive, and this fact suggests the conclusion that the sulfhydryl group of the enzyme is located between the carboxyl affinity points. 5. On the basis of the deduced structure of the succinate-activating center of the enzyme, it is suggested that the enzyme may function by oscillating between the EnSH and EnS· forms, rather than by a thiol-disulfide equilibrium.     

INTRACELLULAR LOCALIZATION OF ENZYMES IN SPLEEN : I. REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE CYTOCHROMEc REDUCTASE, CYTOCHROMEc OXIDASE, AND SUCCINIC DEHYDROGENASE IN THE RAT AND GUINEA PIG

1. The intracellular distribution of nitrogen, DPNH cytochrome c reductase, succinic dehydrogenase, and cytochrome c oxidase has been studied in fractions derived by differential centrifugation from rat and guinea pig spleen homogenates. 2. In the spleens of each species, the nuclear fraction accounted for 40 to 50 per cent of the total nitrogen content of the homogenate, and the mitochondrial, microsome, and supernatant fractions contained about 8, 12, and 30 per cent of the total nitrogen, respectively. 3. Per mg. of nitrogen, DPNH cytochrome c reductase was concentrated in the mitochondria and microsomes of both rat and guinea pig spleens. Seventy per cent of the total DPNH cytochrome c reductase activity was recovered in these two fractions. The reductase activity associated with the nuclear fraction was lowered markedly by isolating nuclei from rat spleens with the sucrose-CaCl₂ layering technique. The lowered activity was accompanied by the recovery of about 90 per cent of the homogenate DNA in the isolated nuclei, indicating that little, if any, of the reductase is present in spleen cell nuclei. 4. Per mg. of nitrogen, succinic dehydrogenase was concentrated about 10-fold in the mitochondria of rat spleen, and 65 per cent of the total activity was recovered in this fraction. 5. Cytochrome c oxidase was concentrated, per mg. of nitrogen, in the mitochondria of both rat and guinea pig spleens. The activity associated with the nuclear fraction was greatly diminished when this fraction was isolated from rat spleens by the sucrose-CaCl₂ layering technique. Only 50 to 70 per cent of the total cytochrome c oxidase activity of the original homogenates was recovered among the four fractions from both rat and guinea pig spleens, while the specific activities of reconstructed homogenates were only 55 to 75 per cent of those of the original whole homogenates. This was in contrast to the results with DPNH cytochrome c reductase and succinic dehydrogenase where the recovery of total enzyme activity approached 100 per cent, and the specific activities of reconstructed homogenates equalled those of the original homogenates. The recovery of cytochrome c oxidase was greatly improved when only the nuclei were separated from rat spleen homogenates. 6. Data were presented comparing the concentrations (ratio of activity per mg. of nitrogen of the fraction to activity per mg. of nitrogen of the homogenate) of DPNH cytochrome c reductase in mitochondria and microsomes derived from different organs of different animals. 7. Data were presented comparing the activities per mg. of nitrogen of DPNH cytochrome c reductase in homogenates from several organs of various animals.     

THE DEMONSTRATION OF THE SUCCINIC DEHYDROGENASE SYSTEM IN BACILLUS SUBTILIS USING TETRANITRO-BLUE TETRAZOLIUM COMBINED WITH TECHNIQUES OF ELECTRON MICROSCOPY

Activity of the succinic dehydrogenase system was studied in Bacillus subtilis utilizing combined techniques of cytochemistry and electron microscopy. Organisms were incubated in a medium containing tetranitro-blue tetrazolium (TNBT) which served as an electron acceptor. Enzymatic activity, as evidenced by deposition of TNBT-formazan, was found on membranous organelles associated with the cytoplasmic membrane and septal plasma membrane, the nuclear area, and the plasma membrane. Flagella, ~190 A in diameter, with thorn-like projections protruded through the cell wall. Tangential-oblique sections of the cell wall showed many pores ~220 A in diameter with a center-to-center spacing of ~450 A.     

INHIBITION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IN MAMMALIAN NERVE BY IODOACETIC ACID

Abstract— Cat sciatic nerves were exposed to iodoacetate for a period of 5–10 min and after washing out the iodoacetate, the enzymes, glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating); EC 1.2.1.12) and lactate dehydrogenase ( l -lactate: NAD oxidoreductase; EC 1.1.1.27) were extracted from the high-speed supernatant fraction of nerve homogenates. Concentrations of iodoacetate as low as 2.5 m m could completely block activity of glyceraldehyde-3-phosphate dehydrogenase but had no effect on lactate dehydrogenase. These findings are in accord with the classical concept shown earlier for muscle that iodoacetate blocks glycolysis by its action on glyceraldehyde-3-phosphate dehydrogenase. A complete block of activity of the enzyme was found after treatment with 2 to 5 m m -iodoacetate for a period of 10 min and such blocks were irreversible for at least 3 h. Glyceraldehyde-3-phosphate dehydrogenase activity was NAD specific, with NADP unable to substitute for NAD. The results are discussed in relation to the effect of iodoacetate in blocking glycolysis and in turn the fast axoplasmic transport of materials in mammalian nerve.     

INHIBITION OF GABA TRANSAMINASE ACTIVITY BY 4-AMINOTETROLIC ACID

Abstract— The influence of the following acetylenic analogues of GABA on GABA-metabolizing enzymes was studied in vitro : 4-amino-, 4-morpholino-, 4-piperazino-, 4-piperidino- and 4-pyrrolidinotetrolic acid. 4-Aminotetrolic acid was a linear competitive inhibitor of GABA transaminase activity in extracts of rat cerebral mitochondria and a linear noncompetitive inhibitor of this enzyme activity in extracts of P. fluorescens when activity was measured with GABA as the variable substrate. From these results it was calculated that the dissociation constants for the binding of 4-aminotetrolic acid to the pyridoxal form of these enzymes are approx. 1 mM. The other substituted tetrolic acids did not influence either transaminase activity under the conditions studied. None of the substituted tetrolic acids influenced the L-glutamic acid decarboxylase activity in extracts of rat cerebral cortex and of E. coli .