SUBCELLULAR DISTRIBUTION AND SOME PROPERTIES OF ACETYL-COENZYME A HYDROLASE IN THE BRAIN

—The detailed subcellular distribution and some properties of acetyl-CoA hydrolase were studied in the rat brain. The brain homogenate (S₁) hydrolysed acetyl-CoA at a rate of approx 2·3 nmol/min/mg of protein at 37°C. The total activity of acetyl-CoA hydrolase was distributed in the following order: soluble > mitochondrial > microsomal, synaptosomal > myelin fraction. The order of the specific activity of the enzyme was: soluble, microsomal > mitochondrial > synaptosomal > myelin fraction. The synaptic vesicle fraction (D) had relatively high specific activity among the intraterminal particulate fractions, having two or three times higher specific activity than that of the synaptic cytoplasmic membrane fraction (F or G). Attempts to de-occlude acetyl-CoA hydrolase in the particulate fraction showed that only the enzyme activity in the myelin fraction was increased markedly by the treatment with ether or Triton X-100. Lineweaver-Burk plots gave straight lines for each subcellular fraction and apparent K_m values for acetyl-CoA were between 0·1 and 0·2 mM. Neither diisopropyl fluorophosphate nor physostigmine at the concentration of 0·1 mm inhibited the enzyme activity.     

SOME PROPERTIES OF GLUTAMATE DECARBOXYLASE AND THE CONTENT OF PYRIDOXAL PHOSPHATE IN BRAINS OF THREE VERTEBRATE SPECIES

—Some properties of glutamate decarboxylase (GAD) were studied in the brain of the carp (Carassius auratus), the pigeon (Columbia livia) and the mouse (Mus musculus). The optimum pH for GAD in the three species was 6·3-6·5. In the three species studied, GAD activity of brain homogenates in water was higher than that of homogenates in buffer. The supernatant from homogenates in Triton-X-100 gave an enzyme preparation which showed greater activation by pyridoxal phosphate than those obtained from complete water or buffer homogenates or from the supernatant of Water homogenates. In the absence of pyridoxal phosphate, the activity of carp GAD was considerably lower than that of mouse or pigeon GAD. The addition of pyridoxal phosphate resulted in a much greater activation of carp GAD than that of pigeon or mouse GAD. Pyridoxal phosphate content was also measured in brains of the species studied. The difference between coenzyme levels in carp and mouse was very small in comparison to the difference in GAD activity in the absence of exogenous coenzyme. The pyridoxal phosphate content of pigeon brain was higher than that of the other two species.     

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.     

Soluble acid phosphatases from the posterior reproductive system of the female housefly, Musca domestica

When the kinetics of the acid phosphatase enzyme system from the posterior reproductive tract of the female housefly, Musca domestica, were studied, enzyme activity was zero order for 2 hr and was temperature-dependent. Orthophosphate release was linearily related to enzyme concentration, and pH optima between pH 3·3 to 3·7, 4·2 to 4·8, and 5·2 to 5·7 were observed. Magnesium and calcium ions were enzyme activators; arsenate, phosphate, fluoride, and hydroxymalonate ions were inhibitors. Sodium azide had little effect. The similarity of activity exhibited on the test substrates indicated that the soluble enzymes corresponded to the non-specific phosphomonoesterases.Disk electrophoresis showed that at least 6 proteins had acid phosphatase activity and were pH-dependent. Also, electrophoresis of extracts of the various structures of the reproductive tract showed that there was a tissue specificity in the distribution of the acid phosphatase isoenzymes.     

The Identification of Fucosterol in the Marine Brown Algae Hizikia fusiformis

The enzyme uridine diphosphate N-acetylglucosamine pyrophosphorylase was purified about 330-fold from an extract of baker’s yeast by the treatment with protamine sulfate and column chromatographies on DEAE-cellulose, hydroxylapatite and Sephadex G–150. The purified enzyme was proved to be homogeneous by disc gel electrophoresis. The molecular weight was determined to be approximately 37,000 by gel filtration. The enzyme had an optimum reactivity in the pH range of 7.5-8.5 and was stable at 4°C in potassium phosphate buffer, pH 7.5, containing 0.1 mm dithiothreitol, but was unstable when stored at ?20°C. The addition of dithiothreitol also increased the thermal stability of enzyme. The enzyme was specific for UDP-N-acetylglucosamine as substrate, and none of the other sugar nucleotides could serve as nucleotide substrate. The estimated values of Km were 6.1 × 10^?3 m for UDP-N-acetylglucosamine and 5.0 × 10^?3 m for inorganic pyrophosphate. The enzyme required some divalent cations for activity. Magnesium ion was the most effective among the cations tested. The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol.     

GABA-TRANSAMINASES OF HUMAN BRAIN AND PERIPHERAL TISSUES—KINETIC AND MOLECULAR PROPERTIES

Abstract— Kinetic experiments with 4-aminobutyrate-2-ketoglutarate transaminase (GABA-T), partially purified from human brain tissue, supported a Bi Bi Ping-Pong type of enzyme mechanism in which the enzyme oscillates between forms bound to pyridoxal phosphate and pyridoxamine phosphate. Extrapolated K _m values were 0.31 m m for γ-aminobutyrate, 0.16 m m for α-ketoglutarate, and 3.8 μ m for pyridoxal phosphate. Very similar kinetic parameters were observed with rat brain enzyme. Apparent molecular weight of human GABA-T by gel filtration was 70,000 ± 3000. Electrofucusing experiments indicated a single ionic form with isoelectric pH = 5.7. Enzyme activity was inhibited by Tris, halides, cadmium and cupric ions, and known GABA-T inhibitors.
GABA-transaminating enzymes isolated from human kidney and liver were found to be similar to the brain enzyme with respect to substrate affinities, cofactor requirements, isoelectric pH values, molecular weights, and response to inhibitors.     

REGIONAL DISTRIBUTION OF GLUTAMIC ACID DECARBOXYLASE IN THE DEVELOPING BRAIN OF THE PYRIDOXINE-DEFICIENT RAT

—Glutamic acid decarboxylase was determined in seven brain regions: hypo-thalamus; midbrain; thalamus; corpus striatum; cerebral cortex-hippocampus; medulla-pons; and cerebellum, of suckling rats subjected to Vitamin B₆ deficiency for 2 weeks from birth; of adult rats subjected to the deficiency for 5 weeks and of their respective controls. Large regional variations in the enzyme activity were found in brains of both adult and suckling control rats. The activity of the enzyme (assayed without pyridoxal phosphate) and its saturation with endogenous cofactor were markedly reduced in all brain regions of both suckling and adult pyridoxine-deficient rats. The apoenzyme (activity assayed with pyridoxal phosphate), in adult rat brain, showed no change with the deficiency in all regions except in the cerebellum where it increased slightly. In pyridoxine-deficient suckling rat brain, the apoenzyme increased substantially in all regions suggesting a process of enzyme induction. The increase in apoenzyme varied from region to region.     

Solubilization,purification, and properties of rabbit brain hexokinase

More than 90% of the total hexokinase activity in rabbit brain was found to be associated with the mitochondrial fraction. The participate enzyme was solubilized in a relatively specific way by glucose 6-phosphate and Triton X-100 and purified to apparent homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and affinity chromatography. The solubilized hexokinase activity has been purified 700-fold in 48% yield with a specific activity of 165 units/mg of protein. The molecular weight was found to be approximately 100,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.3 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses with different affinities. Mg · ATP, among the nucleotide substrates, was the most effective as a phosphate donor. The present results indicate considerable similarity between this enzyme and the other mammalian type I hexokinases.     

Metabolisms of Nucleosides in Bacteria

The mechanism of trans-N-ribosylation in Corynebacterium sepedonicum was investigated. Using the DEAE-cellulose colum chromatography, this enzyme activity was divided into two fractions. One cleaved uridine to uracil and ribose phosphate, and the other decomposed inosine into hypoxanthine and ribose phosphate, in the presence of inorganic phosphate. The ribose phosphate was isolated and crystallized.

Several analytical data indicated that the ribose phosphate was ribose-1-phosphate. These two enzyme fractions catalyzed the formation of nucleosides from ribose-1-phosphate and bases.

Most of bacteria, which had the activity to transfer N-ribosyl group between purine and pyrimidine, could synthesize the nucleoside from base and ribose-1-phosphate.     

Sucrose biosynthesis in Dunaliella

Sucrose phosphate synthetase (EC 2.4.1.14) is the key enzyme for sucrose synthesis in Dunaliella tertiolecta. It has been partially purified and characterized. The enzyme contains one binding site for uridine diphosphoglucose and two binding sites for fructose-6-phosphate; it is allosterically controlled by fructose-6-phosphate. Inorganic phosphate stimulates the enzymic activity, particularly in the presence of higher concentrations of fructose-6-phosphate. Sucrose phosphate synthetase is not halophilic or halotolerant. The temperature dependence of the enzymic activity cannot fully explain the observed increase in sucrose synthesis in Dunaliella by elevated temperature.Abbreviations F-6-P fructose 6-phosphate - UDP uridine biphosphate - UDPG uridine biphosphoglucose     

Uridine diphosphate D-glucose dehydrogenase of Aerobacter aerogenes

Uridine diphosphate d-glucose dehydrogenase (EC 1.1.1.22) from Aerobacter aerogenes has been partially purified and its properties have been investigated. The molecular weight of the enzyme is between 70,000 and 100,000. Uridine diphosphate d-glucose is a substrate; the diphosphoglucose derivatives of adenosine, cytidine, guanosine, and thymidine are not substrates. Nicotinamide adenine dinucleotide (NAD), but not nicotinamide adenine dinucleotide phosphate, is active as hydrogen acceptor. The pH optimum is between 9.4 and 9.7; the K(m) is 0.6 mm for uridine diphosphate d-glucose and 0.06 mm for NAD. Inhibition of the enzyme by uridine diphosphate d-xylose is noncooperative and of mixed type; the K(i) is 0.08 mm. Thus, uridine diphosphate d-glucose dehydrogenase from A. aerogenes differs from the enzyme from mammalian liver, higher plants, and Cryptococcus laurentii, in which uridine diphosphate d-xylose functions as a cooperative, allosteric feedback inhibitor.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

In vitro transformation of dehydroepiandrosterone or its sulphate into androstenediol or its sulphate by rat brain and blood preparations

Abstract— –Enzymic transformation of [4-¹⁴C]dehydroepiandrosterone or [4-¹⁴C]dehydro-epiandrosterone sulphate to androstenediol or its sulphate occurred when incubated with a microsomal preparation of rat brain or a whole rat blood homogenate. The brain enzyme which appeared to cause this transformation had a pH optimum at 60, was NADPH₂-dependent, and had an apparent K_m of 4·6 × 10^?6m . When the subcellular fractions of rat brain were compared for transformation, microsomes had the highest specific activity, followed by the cytosol. The crude nuclear and mitochondrial fractions had no significant activity. The level of enzymic activity in the brain microsomes increased from that for rats sacrificed at 7 days of postnatal age to a maximum for rats sacrificed at 1 month of age; then the activity appeared to level off in rats older than 1 month. Microsomes obtained from the cerebellum had the highest specific activity in comparison to that obtained from the cerebral cortex, the diencephalon, and the brain stem. The incubated preparations of rat brain also converted dehydroepiandrosterone sulphate to androstenediol sulphate without hydrolysis. The enzyme in rat blood which was similar to that in the brain was also partially characterized. The blood enzyme had a pH optimum at 6–5, was nearly exclusively present in erythrocytes, was also NADPH₂-dependent, and had an apparent K_m of 2·7 × 10^?4m . The developmental pattern of the blood enzyme specific activity was similar to that of the rat brain enzyme. Upon haemolysis, most activity was recovered in the haemolysate.     

METABOLIC PARAMETERS OF ONTOGENESIS OF ELECTRICAL ACTIVITY IN THE BRAIN. SODIUM-POTASSIUM ACTIVATED ADENOSINE TRIPHOSPHATASE IN DEVELOPING CHICK EMBRYO BRAIN

—(1) The activity of the Na-K ATPase in the particulate fraction of the chick embryo brain has been assayed at different stages of development with the objective of finding whether or not changes in the activity of this enzyme bear any relation to the maturation of spontaneous and evoked electrical activity of the growing chick brain. (2) The specific activity of the enzyme is low on day 6 and it rises rapidly between days 10 and 12, at which time it attains a plateau and remains essentially unchanged from day 12 until day 20. Experimental evidence rules out the possible presence of an inhibitor of the enzyme in 8-day-old brain homogenates, suggesting that these developmental changes in the activity of the enzyme may represent new synthesis of enzyme rather than its activation. The period between days 10 and 12 does not represent a unique stage of general protein synthesis. (3) The chick brain particulate enzyme has an optimum activity at pH 7·4 and at 37°. It is optimally activated by a Na⁺ concentration of 100mm and K⁺ concentration of 20 mm . The enzyme is inhibited by ouabain and Ca²⁺. (4) The results have been discussed.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.     

Creatine kinase of heart mitochondria: Changes in its kinetic properties induced by coupling to oxidative phosphorylation

A complete kinetic analysis of the forward mitochondrial creatine kinase reaction was conducted to define the mechanism for its rate enhancement when coupled to oxidative phosphorylation. Two experimental systems were employed. In the first, ATP was produced by oxidative phosphorylation. In the second, heart mitochondria were pretreated with rotenone and oligomycin, and ATP was regenerated by a phosphoenolpyruvate-pyruvate kinase system. Product inhibition studies showed that oxidative phosphorylation did not effect the binding of creatine phosphate to the enzyme. Creatine phosphate interacted competitively with both ATP and creatine, and the E · MgATP · CrP dead-end complex was not readily detected. In a similar manner, the dissociation constants for creatine were not influenced by the source of ATP: K_ib = 29 mm; K_b = 5.3 mM, and the maximum velocity of the reaction was unchanged: V₁ = 1 μmol/ min/mg. Slight differences were noted for the dissociation constant (K_ia) of MgATP from the binary enzyme complex, E · MgATP. The values were 0.75 and 0.29 mm in the absence and presence of respiration. However, a 10-fold decrease in the steady-state dissociation constant (K_a) of MgATP from the ternary complex, E · MgATP · creatine, was documented: 0.15 mm with exogenous ATP and 0.014 mm with oxidative phosphorylation. Since K_ia × K_b does not equal K_a × K_ib under respiring conditions, the enzyme appears to be altered from its normal rapid-equilibrium random binding kinetics to some other mechanism by its coupling to oxidative phosphorylation.     

Solubilization and properties of polyprenyl phosphate: GDP-D-mannose mannosyl transferase

A soluble enzyme which catalyzes the formation of dolichyl β-d-mannosyl phosphate has been prepared from encysting cultures of Acanthamoeba castellanii. The enzyme is relatively specific for GDP-d-mannose in that GDP-d-glucose and various uridine nucleotides do not serve as substrates. Uridine diphosphate d-glucose is not an inhibitor at 100-fold molar excess concentration, but GDP-d-glucose, GDP, and GMP do inhibit the reaction at relatively high concentrations. The apparent K_m for GDP-d-mannose is approximately 0.25 μm and that for dolichyl phosphate is approximately 3.3 μm. The enzyme has a pH optimum of 7.0, a temperature optimum of 27 °C, and requires a divalent cation. Magnesium, cobalt, and manganese salts will serve as cofactors but maximum activity is produced by Mn²⁺. No loss of activity is evident after storage for 2 weeks at ?70 °C, but half the activity was lost within 3 days at 0 °C, and a third of the activity was lost within 2 weeks at ?20 °C.     

Non-allosteric regulation of the uridine kinase from seeds of Zea mays.

Uridine kinase (ATP: uridine 5'-phosphotransferase, EC 2.7.1.48) has been partially purified from ungerminated hybrid corn seed. It is associated with a soluble high molecular weight fraction from which it apparently cannot be dissociated without loss of activity. The stability of the enzyme is enhanced by the addition of dithiothreitol, glycerol and nucleotide substrate. The nucleoside specificity of the enzyme is limited to nucleosides containing pyrimidine and ribose moieties, such as uridine and cytidine. High concentrations of nucleosides cause substrate inhibition, however. The Km values for uridine and cytidine are 53 muM and 125 muM, respectively, and under subsaturating conditions uridine is phosphorylated about five times faster than cytidine. The reaction follows an ordered Bi Bi kinetic pattern, with ATP and ADP in competition for the free form of the enzyme. Purine, but not pyrimidine, nucleoside triphosphates serve as phosphate donors without regard to the sugar moiety. However, all of these triphosphates appear to compete for the same site on the enzyme. (Km ATP equals 590 muM, Km (app) GTP equals 61 muM, and CTP and UTP are linear competitive inhibitors against ATP, with Ki values of 60 muM and 240 muM, respectively.) Therefore, end product control of uridine kinase apparently does not involve allosteric sites, but instead is envisioned as simple competition between relatively effective or ineffective phosphate donors for a position on the enzyme.     

Yersinia pseudotuberculosis nucleoside-kinase

Enzyme capable of catalyzing the phosphorylation of thymidine and uridine was isolated from Y. pseudotuberculosis cells by fractionation with the use of ammonium sulfate, ion exchange and affinity chromatography. The degree of purification of thymidine- and uridine-kinase was approximately 350 times, and at all stages of isolation the activity of both nucleoside-kinases was detected in the same peaks. The purified enzyme was capable of the phosphorylation of thymidine and uridine at temperatures of 8-10 degrees C to 50 degrees C and exhibited the maximum enzymatic activity at pH 8-8.5 and 45 degrees C in the presence of 0.5-1.0 mM MgCl2 and 2 mM ATP. The enzyme was found to have no strict substrate specificity and transferred the phosphate group from ATP to radiolabeled thymidine, uridine and desoxycytidine with different effectiveness, but did not use thymidine-monophosphate as phosphate acceptor.     

Assay and subcellular localization of the arylsulphatases in rat brain

—The properties and subcellular localization of type I (nitrophenyl) and type II (nitrocatechol) arylsulphatases were investigated in brain tissue of the rat, and optimal assay conditions were established. Sulphate, phosphate and sulphite ions inhibited the nitrocatechol sulphatases; nitrophenyl sulphatase was inhibited only by sulphite. The presence of latent enzyme activity was demonstrated for the nitrocatechol sulphatases, beta-glucuronidase, and beta-glycerophosphatase in rat and mouse brain homogenates. These hydrolases were highly sensitive to mechanical and osmotic damage; and Triton X-100 was very effective in releasing their latent (bound) activities, a finding suggestive of a lysosomal localization. Activity of nitrophenyl sulphatase was unaffected by osmotic changes or Triton X-100, characteristics suggesting a membranous association for this enzyme. Total activity of nitrophenyl sulphatase was approximately twice as great in canine gray matter as in canine white matter; the converse obtained for beta-glucuronidase activity. Values for total enzymic activity of the nitrocatechol sulphatases in canine white and gray matter were similar. Fractionation of homogenates from rat brain by differential centrifugations and separation of crude mitochondrial fractions by sucrose density gradient centrifugations revealed the following: (1) most of the nitrocatechol sulphatase activity (93 per cent) and all of the nitrophenyl sulphatase activity were sedimentable; (2) crude mitochondrial fractions exhibited the highest relative specific activity (RSA = 1·38) for the nitrocatechol sulphatases, whereas microsomal fractions displayed the highest RSA for nitrophenyl sulphatase (1·89); (3) the lightest fraction (A + B) and the densest fraction (E) from the sucrose density gradient contained most of the activity for both the type I and type II arylsulphatases, whereas the RSA of cytochrome oxidase was greatest in the intermediate density regions (fractions C and D); (4) the highest RSA for beta-glucuronidase and beta-glycerophosphatase occurred in gradient fraction C; (5) appreciable activity of beta-glycerophosphatase was found in a nerve ending fraction (M₃). It is suggested that the hydrolases in heterogeneous tissue like brain might be associated with lysosomal particles of differing enzyme compositions and varying populations, and that the data on distribution lend credence to the concept of bimodal and possible trimodal particle affinity for the hydrolases of brain tissues.