GLYCINE DECARBOXYLATION IN THE CENTRAL NERVOUS SYSTEM

Abstract— The regional and subcellular distribution of the glycine decarboxylation which occurs in the presence of tetrahydrofolate, NAD and pyridoxal phosphate, has been measured in CNS tissue of cat, sheep and rat. The activity appeared similar to that of liver. It was located within mitochondria, and distributed regionally and subcellularly in the same manner as succinate dehydrogenase, a mitochondrial marker. Activity was low in pons, medulla and spinal cord, and was not affected by a number of drugs, some of which excite and some of which depress the activity of the CNS. All evidence suggests that glycine decarboxylation plays no direct role in glycine inhibitory transmission.     

POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2-OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10-day-old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1- and 10-day-old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2-oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10-day-old rats suggests that the activity of this enzyme reflects cell growth rate.     

The oxidation of d- and l-glycerate by rat liver

1. The interconversion of hydroxypyruvate and l-glycerate in the presence of NAD and rat-liver l-lactate dehydrogenase has been demonstrated. Michaelis constants for these substrates together with an equilibrium constant have been determined and compared with those for pyruvate and l-lactate. 2. The presence of d-glycerate dehydrogenase in rat liver has been confirmed and the enzyme has been purified 16–20-fold from the supernatant fraction of a homogenate, when it is free of l-lactate dehydrogenase, with a 23–29% recovery. The enzyme catalyses the interconversion of hydroxypyruvate and d-glycerate in the presence of either NAD or NADP with almost equal efficiency. d-Glycerate dehydrogenase also catalyses the reduction of glyoxylate, but is distinct from l-lactate dehydrogenase in that it fails to act on pyruvate, d-lactate or l-lactate. The enzyme is strongly dependent on free thiol groups, as shown by inhibition with p-chloromercuribenzoate, and in the presence of sodium chloride the reduction of hydroxypyruvate is activated. Michaelis constants for these substrates of d-glycerate dehydrogenase and an equilibrium constant for the NAD-catalysed reaction have been calculated. 3. An explanation for the lowered V_max. with d-glycerate as compared with dl-glycerate for the rabbit-kidney d-α-hydroxy acid dehydrogenase has been proposed.     

THE HISTOCHEMICAL DEMONSTRATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ACTIVITY

A histochemical method for demonstration of glyceraldehyde-3-phosphate dehydrogenation by tissues is described. The method utilizes Nitro BT as an indicator, glyceraldehyde-3-phosphate obtained from hydrolysis of commercially obtainable glyceraldehyde-3-phosphate diethylacetal (monobarium salt) as substrate, and (ethylenediamine)tetraacetic acid acid disodium as an activating agent in a medium buffered to pH 7.2 by 0.2 M sodium phosphate. The heat lability, substrate and coenzyme specificity, and sulfhydryl and phosphate dependence of the tissue component catalyzing this reaction indicate that glyceraldehyde-3-phosphate dehydrogenase activity is being demonstrated. The disparity between the known pH optimum of this enzyme and that determined histochemically, and the anomalous histochemical localization to mitochondria of this enzyme which has been found in the soluble fraction by differential centrifugation, are thought to result from the diaphorase dependence of the tetrazolium methods and to emphasize the need for caution in the interpretation of histochemically determined intracellular localization of dehydrogenating enzymes. The evidence gathered by previous workers concerning the feasibility of demonstrating specific dehydrogenases with Nitro BT, and the correspondence of the distribution of glyceraldehyde-3-phosphate dehydrogenase determined histochemically with available quantitative data, suggest that at the cellular level the histochemical results accurately reflect the distribution of this enzyme.     

STUDIES ON THE PROPERTIES OF RETINAL ALCOHOL DEHYDROGENASE FROM THE RAT

An NAD-dependent alcohol dehydrogenase (alcohol:NAD oxidoreductase; EC 1.1.1.1) has been isolated and partially purified from the retinal cytosol of the rat. Its substrate specificity and sensitivity to inhibitors of hepatic alcohol dehydrogenase have been investigated. Ethanol, 1-propanol and 1-butanol served as substrates for this enzyme but the K_m values were more than 100-fold higher than those reported for hepatic alcohol dehydrogenase. Methanol and retinol were unreactive with this alcohol dehydrogenase. Inhibition by pyrazole was observed but the K_t was about 100-fold higher than the value observed for hepatic alcohol dehydrogenase. n-Butyraldoxime inhibited retinal alcohol dehydrogenase with a K_t of 2 μM, a value which approximates its K_t for hepatic alcohol dehydrogenase. 1, 10-Phenanthroline was ineffective as an inhibitor. Oxidation of retinol was observed in retinal homogenates in the presence of NADP but no inhibition was observed with ethanol, methanol or pyrazole. We conclude that oxidation of retinol is not catalysed by soluble retinal alcohol dehydrogenase.     

Effects of 2,3-diphosphoglyceric acid on the human erythrocyte membrane phosphorylation system

The effect of 2,3-diphosphoglycerate (2,3-P2-glycerate) on the phosphorylation of spectrin in solution by purified membrane cyclic AMP-independent protein kinase and in membrane preparations by the endogenous kinase was investigated. 2,3-P2-Glycerate inhibited spectrin phosphorylation both in solution and in the intact membrane. Kinetic analyses showed that 2,3-P2-glycerate had no effect on the Km for ATP but appeared to lower the Vmax of the reaction. When the effect of 2,3-P2-glycerate was examined in the presence of varying concentrations of spectrin, competitive inhibition kinetics were obtained. Interestingly, low concentrations of 2,3-P2-glycerate were found to effect the release of the membrane kinase from erythrocyte membranes. This release reaction may be related to the ability of 2,3-P2-glycerate to interfere with the interaction between the kinase and spectrin. The data suggest the possibility that the kinase may be bound to spectrin in the erythrocyte membrane. 2,3-P2-glycerate also caused the solubilization of 3-phosphoglyceraldehyde dehydrogenase, but not of cyclic AMP-dependent protein kinase. Taken together, our data indicate that 2,3-P2-glycerate may have a regulatory role in membrane protein phosphorylation and also may regulate the extent of association of the kinase with the membrane.     

DISTRIBUTION OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE TRANSAMINASE IN SEVERAL AREAS OF THE CENTRAL NERVOUS SYSTEM OF THE RAT

—The regional distributions of serine hydroxymethyltransferase (SHMT) and glycine transaminase (GT) have been determined in five areas of the CNS of the rat. The SHMT activity per mg protein varied in these areas in the following order: medulia-pons and spinal cord > cerebellum > midbrain > telencephalon. The GT activity per mg protein was essentially the same in the four brain areas, whereas, in the spinal cord it was lower. The activity of GT did not correlate with the glycine content (r=?0.45. P > 0.05). However, SHMT activity per mg protein was correlated with the glycine content in four regions (the telencephalon, midbrain, medulla-pons and spinal cord; r= 0.997, P < 0.05). When the activity of SHMT was expressed per relative number of mitochondria, the enzyme levels were correlated with the glycine content in all five areas (r= 0.952, P < 0.05). The distribution of SHMT was determined in the primary subcellular fractions of the CNS. The SHMT activity in these areas of the CNS appeared to be located predominately in paniculate structures, while only 1 to 4 per cent was found in the soluble fraction. The crude nuclear (P₁) and the crude mitochondrial (P₂) fractions contained 90–97 per cent of the activity. Subfractionation of P₂ pellets obtained from the telencephalon, medulla-pons and spinal cord indicated the SHMT activity was localized in both ‘free’ and occluded mitochondria.     

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 REGIONAL AND SUBCELLULAR DISTRIBUTION OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Catechol-O-methyl transferase (COMT) activities determined in different regions of rat brain showed small variations. Highest activities were found in the hypothalamus and corpora quadrigemina, and lowest activities in the hippocampus and corpus striatum. The regional distribution of COMT was thus at variance with the distribution of DOPA decar- boxylase in this study and with the distribution of catecholamines and tyrosine hydroxylase reported in the literature. Determinations of the subcellular distribution of COMT in rat forebrain showed that 50 per cent of the activity was recovered in the high speed supernatant fluid and about 33 per cent in the crude mitochondrial fraction. Further separation of the latter by discontinuous sucrose gradients showed that the particulate COMT was found in the synaptosomal fraction in an occluded form. Full enzyme activity was only obtained after treatment with a detergent or after resuspension in water. After hypo-osmotic rupture of the crude mitochondrial fraction, COMT was recovered in the cytoplasmic fraction. The subcellular distribution of COMT was very similar to the ones of lactate dehydrogenase and DOPA decarboxylase. The proportions of soluble COMT obtained from homogenates of various regions of the brain differed from that of choline acetyl transferase and DOPA decarboxylase but were similar to that of lactate dehydrogenase. In conclusion, COMT is a cytoplasmic enzyme almost evenly distributed in the CNS. Its distribution does not resemble the distributions of the catecholamines or of the enzymes participating in the synthesis of catecholamines.     

Sensitive Immunoassay Shows Selective Association of Peripheral and Integral Membrane Proteins of the Inhibitory Glycine Receptor Complex

The inhibitory glycine receptor of mammalian spinal cord is a ligand-gated chloride channel that, on affinity purification, contains two subunits of 48-kilodalton (kD) and 58-kD molecular mass in addition to an associated 93-kD protein. Ligand-binding 48-kD subunit and 93-kD protein were quantified in the CNS of the adult rat using a newly developed dot receptor assay (detection limit less than or equal to 1 fmol/assay) which employs monoclonal antibodies specific for glycine receptor polypeptides. The 93-kD protein was found to codistribute at a fixed stoichiometry with the 48-kD subunit throughout the CNS of the rat. Moreover, the 93-kD protein cofractionated with the ligand-binding subunit on solubilization and affinity chromatography or immunoprecipitation. However, both proteins were separated on sucrose gradient centrifugation of detergent extracts of spinal cord membranes in accord with earlier observations on purified receptor. These data prove that the 93-kD polypeptide is selectively associated with the membrane core of the strychnine-sensitive glycine receptor. The regional distribution of glycine receptor polypeptides was also determined in the CNS of the spastic rat mutant. In contrast to hereditary spasticity in mouse and cattle, no reduction of glycine receptors was found in the spastic rat.     

CHANGES IN THE LACTATE AND MALATE DEHYDROGENASE ISOENZYME PATTERNS OF CHICKEN EMBRYO BRAIN AND RETINA

Abstract— Lactate dehydrogenase and malate dehydrogenase isoenzyme patterns of chicken brain and retina have been investigated by cellulose acetate electrophoresis, during embryonic and post-hatching development. The proportion of M-type lactate dehydrogenase subunits decreases significantly in brain and retina with development. A marked increase in the H-type subunits is observed in retina. Lactate dehydrogenase isoenzyme distribution appears to change in both organs in parallel with the metabolic changes of differentiation.
Malate dehydrogenase isoenzyme patterns do not reveal any consistent change of the ratio between the mitochondrial and cytoplasmic forms.     

THE REGULATION OF PYRUVATE DEHYDROGENASE IN BRAIN IN VIVO

—The activity of pyruvate dehydrogenase in the brains of mice frozen in liquid nitrogen was 14·0 nmol/min per mg protein. It rose to 23·8 nmol/min per mg protein after incubation of the brain homogenate with 10mm -MgCl₂ to activate (dephosphorylate) the enzyme, indicating that approx 60% of the enzyme was originally in the active form. Treatment with amobarbital or pentobarbital halved the proportion of pyruvate dehydrogenase in the active form. The proportion of pyruvate dehydrogenase in the active form increased during ischemia, activation being complete within one min. Anesthesia with amobarbital slowed the activation during ischemia but did not alter the total amount of pyruvate dehydrogenase activity. The concentration of ATP, the ATP/ADP ratio and the adenylate energy charge increased as the proportion of pyruvate dehydrogenase in the active form decreased during barbiturate anesthesia, and they decreased as the proportion of pyruvate dehydrogenase in the active form increased during ischemia. After treatment with insulin, the proportion of pyruvate dehydrogenase in the active form increased by 30%. but the energy charge did not change. Treatment of mice with ether, morphine, ethanol, or diazepam did not change the proportion of pyruvate dehydrogenase in the active form although these treatments have been reported to alter pyruvate oxidation in brain in vivo. Treatments which altered pyruvate oxidation in the brain did not consistently alter the proportion of pyruvate dehydrogenase in the active form, unless they also altered energy charge.     

REGIONAL DISTRIBUTION AND PROPERTIES OF THE GLYCINE CLEAVAGE SYSTEM WITHIN THE CENTRAL NERVOUS SYSTEM OF THE RAT: EVIDENCE FOR AN ENDOGENOUS INHIBITOR DURING IN VITRO ASSAY

—The activity of the glycine cleavage system (GCS) was determined in homogenates from five specific regions of the rat CNS (telencephalon, midbrain, cerebellum, medulla-pons, and spinal cord). An inverse trend was noted between the glycine content and the specific activity of the GCS in the regions. A 25-fold range in the enzyme activities was found between the telencephalon (highest) and the spinal cord (lowest). The properties of the GCS activity in CNS homogenates agreed with those properties previously described for this system in partially purified preparations of liver and brain mitochondria (Kikuchi , 1973; Bruin et al., 1973). Within the CNS homogenates, the liberation of CO₂ from the carboxyl carbon of glycine was quantitatively coupled to the formation of serine. The presence of an endogenous inhibitor(s) within neural tissues was suggested by the non-additivity of the activities when homogenates from the various regions were combined. Moreover, homogenates of CNS tissue inhibited the GCS activity of liver homogenates, and an inverse relationship was found between the level of GCS activity in a given region of the CNS and its ability to inhibit the GCS activity of liver homogenates. This inhibition of liver activity was greatest when liver was incubated with homogenates of spinal cord (86%) and lowest when incubated with homogenates of telencephalon (20%). Because of this endogenous inhibition, the apparent activity of the GCS measured in vitro may not reflect the contribution of this enzyme system in the metabolism of glycine in vivo. Although the significance of this inhibition is not known, a possible role is discussed for the regulation of the levels in glycine and one-carbon pools within the CNS.     

HISTOCHEMICAL DEMONSTRATION OF DEHYDROGENASE ACTIVITY IN THE CELLS OF NORMAL HUMAN BLOOD AND BONE MARROW

Endogenous and succinic dehydrogenase activity was demonstrated in the living cells of normal human blood and bone marrow using a buffered nitro BT-succinate incubating solution. With this technique dehydrogenase activity was localized primarily in the granular leukocytes and the sites of enzymatic activity appeared to be non-mitochondrial. The addition of a non-ionic surface active agent to the incubating solution resulted in marked differences in the cellular and intracellular localization of dehydrogenase activity. With this method it was possible to demonstrate dehydrogenase activity in the mitochondria of most of the formed elements of the blood and bone marrow, including developing granulocytes and erythroid cells, agranulocytes, and blood platelets. Mature erythrocytes also exhibited a minimal dehydrogenase reaction with this procedure. This investigation indicated that in order adequately to demonstrate and evaluate dehydrogenase activity in the cells of the blood and bone marrow it was necessary to have increased cellular and mitochondrial permeability, as well as partially viable cells with an intact dehydrogenase system.     

Enzyme Regulation in C(4) Photosynthesis : PURIFICATION AND PROPERTIES OF THIOREDOXIN-LINKED NADP-MALATE DEHYDROGENASE FROM CORN LEAVES

NADP-malate dehydrogenase, a light-modulated enzyme of C₄ photosynthesis, was purified to homogeneity from leaves of corn. The pure enzyme was activated by thioredoxin m that was reduced either photochemically (with ferredoxin and ferredoxin-thioredoxin reductase) or chemically (with dithiothreitol). Unactivated corn leaf NADP-malate dehydrogenase had a molecular weight of 50,000 to 60,000 and was chromophorefree. The enzyme appeared to have a high content of serine and glycine and to contain both S—S and SH groups. Consequently, NADP-malate dehydrogenase seems to be capable of undergoing reversible oxidation/reduction during its photoregulation.     

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.     

Studies on avian erythrocyte metabolism. XVI. Accumulation of 2,3-bisphosphoglycerate with shifts in oxygen affinity of chicken erythrocytes

The ability of the chicken erythrocyte to accumulate 2,3-bisphosphoglycerate (2,3-P2-glycerate) and its effect upon the oxygen affinity (P50) of the cell suspensions have been determined. Erythrocytes from chick embryos, which contain 4-6 mM 2,3-P2-glycerate, and from chickens at various ages, which contain 3-4 mM inositol pentakisphosphate but no 2,3-P2-glycerate, were incubated with inosine, pyruvate, and inorganic phosphate. Red blood cells from 20-day chick embryos incubated in Krebs-Ringer, pH 7.45, containing 20 mM inosine and 20 mM pyruvate had an increase in 2,3-P2-glycerate from 4.3 to 11.9 mM after 4 h. Importantly, as 2,3-P2-glycerate concentration increased there was a corresponding increase in P50 of the cell suspension. Further, erythrocytes from 9- and 11-week, and 7-, 14-, 24-, and 28-month-old chickens when incubated similarly with inosine and pyruvate accumulated 2,3-P2-glycerate with corresponding increases in P50 of the cell suspensions. The ability of the red cell to accumulate this compound under the incubation conditions used apparently decreases with age of the bird (e.g., 11.9 mM in the 20-day embryo to 1.1 mM in the 28-month-old chicken after 4 h incubation). Despite the presence of significant amounts of inositol-P5, the accumulation of 2,3-P2-glycerate markedly decreases oxygen affinity of the cell suspensions. The delta P50/mumol increase in 2,3-P2-glycerate in the red cells of the 20-day chick embryo after 4 h incubation is 1.5 Torr; conversely, the delta P50/mumol decrease in 2,3-P2-glycerate in the red cells of the 17-day embryo after 6 h incubation in the presence of sodium bisulfite is 2.8 Torr. The demonstrated ability of the chicken erythrocyte to accumulate 2,3-P2-glycerate in response to certain substrates suggests that regulation of concentration of this compound could contribute significantly to regulation of blood oxygen affinity in birds.     

Regional distribution of putative amino acid neurotransmitters in the CNS of spiders (Arachnida: Araneida)

The regional occurrence of five amino acid neurotransmitters (GABA, taurine, glycine, glutamate, aspartate) was studied in the CNS of five spider families, using a fluorescence microchromatogram method. Clear differences in transmitter distribution were obvious in relation to the phylogenetic standard of the spider families and the CNS regions investigated. For example, high relative amounts of taurine and GABA were typically confined to the protocerebral brain parts, particularly in the Araneidae and Salticidae. Remarkable amounts of glutamate were shown to exist in the lower CNS parts of the Agelenidae and, especially, the Theraphosidae.The results obtained confirm the dominant functional role of the protocerebrum in arthropods.     

DISTRIBUTION AND UPTAKE OF AMINO ACIDS IN VARIOUS REGIONS OF THE CAT BRAIN IN VITRO

—(1) The levels of the free amino acids were determined in five areas of the cat brain. The regional pattern was heterogeneous and fairly characteristic for each compound. (2) The uptakes of α-aminoisobutyric acid, taurine, d -aspartic acid, and l -histidine were measured in incubated slices from 31 regions of the cat CNS. Differences in uptake were found among the various areas; the regional pattern of uptake was different for each amino acid. The initial rate of uptake (5 min incubation) very often paralleled the rate at equilibrium (90 min incubation). (3) The regional correlation between distribution in vivo and uptake in vitro was good for aspartate, less so for histidine, and poor for taurine. (4) It is concluded that regional heterogeneity in exit processes, available energy, cell density, or protein content is unlikely to have decisive influence in determining regional differences in distribution and transport of metabolites; it seems that influx is the most important factor.     

类杆菌分类的脱氢酶系列

国外已建立了4h水解酶色原底物系列,成功地用于厌氧菌的分类与鉴定,鉴于国内的无系列水解底物,我们对厌氧菌系列脱氢酶进行了研究,以便建立一种快速鉴定系列。为此选择类杆菌的参考株作为分类单位,以不同的糖、氨基酸和有机酸的天然底物纸片与细菌完整细胞进行酶学反应,并以四唑盐受氢后以变色效应来指示酶的存在。筛选了18项脱氢底物与色氨酸、尿素一起,组成20项以检测细菌脱氢酶为主的系列,研究发现完整的菌细胞可以较好地表现底物的脱氢酶活性,酶的分布具有菌种特征,这个系列在有氧条件下,30～60min出现明显的可见反应。以4株脆类杆菌参考株进行24次重复试验,其中9项的阳性概率为0.99,7项为0.01,其它4项分别是0.03,0.125,0.5和0.66。尤其胆酸脱氢酶与常规胆汁抑制试验有较好的相关性,有可能取代厌氧菌分类中有要意义的20％胆汁抑制实验。