Content of thiamin phosphate esters in mammalian tissues--an extremely high concentration of thiamin triphosphate in pig skeletal muscle

In relation to a high activity of thiamin diphosphate (TDP) kinase (Koyama, S. et al. (1985) Biochem. Int. 11, 371-380) in the skeletal muscle of pigs and guinea pigs, the content of thiamin phosphate esters in tissues of these animals has been determined by the method of high-performance liquid chromatography. An extremely high concentration of thiamin triphosphate (TTP), 69.2% of the total thiamin (26.1 nmol/g wet weight), was detected in adult pig skeletal muscles. One extreme case contained TTP as 88.7% of the total thiamin (19.6 nmol/g wet weight). TTP in pig skeletal muscle was found solely in cytosol fraction. This is the first report showing an unusually high level of TTP in mammals and may give a clue as to the physiological functions of TTP.     

Cytosolic adenylate kinase catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate

An attempt was made to purify a porcine skeletal muscle enzyme catalyzing the formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP), requiring ATP, Mg2+ and a cofactor (creatine). As the purification proceeded, the reaction requirements for ATP and creatine were lost and then a requirement for ADP was manifested. The activity responsible for TTP synthesis from TDP, ADP, and Mg2+ was found to be copurified with adenylate kinase [EC 2.7.4.3] activity, and was finally purified to a single band on SDS-PAGE. Antiserum obtained against the purified enzyme preparation inhibited both adenylate kinase activity and the TTP-synthesizing activity to exactly the same extent. These results indicate that adenylate kinase catalyzes TTP formation from TDP in vitro.     

Properties of thiamin triphosphate-synthesizing activity of chicken cytosolic adenylate kinase and the effect of adenine nucleotides

Cytosolic adenylate kinase synthesis thiamin triphosphate (TTP) from thiamin diphosphate (TDP) in vitro by a reversible reaction: TDP + ADP Mg2+ in equilibrium TTP + AMP. The backward (TTP----TDP) reaction rate was 3-times faster than the forward (TDP----TTP) reaction rate when all the substrate concentrations were 0.1 mM. This property of TTP-synthesizing activity of the enzyme did not explain the fact that the [TTP]/[TDP] molar ratio determined in chicken white skeletal muscle is 5.0 (Miyoshi, K., Egi, Y., Shioda, T. and Kawasaki, T. (1990) J. Biochem. 108, 267-270). To solve this problem, we have studied the properties of TTP-synthesizing activity of the purified recombinant chicken cytosolic adenylate kinase preparation and the effect of adenine nucleotides, especially of ATP. The backward reaction of the TTP synthesis did not proceed in the presence of 8.8 mM ATP, a physiological concentration in chicken white skeletal muscle, while the forward reaction proceeded at a reduced rate. The [TTP]/[TDP] ratio found after a long incubation period was 3.0 and 0.7, respectively, in the presence and absence of 8.8 mM ATP. These results indicate that the high [TTP]/[TDP] molar ratio found in chicken white muscle was demonstrated in vitro by the purified chicken cytosolic adenylate kinase and support in vivo TTP synthesis by this enzyme.     

Evidence for in vivo synthesis of thiamin triphosphate by cytosolic adenylate kinase in chicken skeletal muscle

We showed previously that cytosolic adenylate kinase (AK1) purified from pig skeletal muscle catalyzes in vitro formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP) and ADP in addition to ATP formation from ADP [Shikata, H. et al. (1989) Biochem. Int. 18, 933-942]. To obtain evidence for in vivo synthesis of TTP by AK1, changes in TTP content and AK1 activity were determined in chicken skeletal muscle during development after hatching. Thiamin phosphate metabolism in chicken skeletal muscle was also studied. i) An extremely high TTP content, 81% of total thiamin (thiamin plus thiamin phosphates), was detected in the white (fast-twitch) muscle of adult normal chicken (5th to 9th month) compared with a relatively high TTP content of 31% in the red (slow-tonic) muscle. Since approximately equivalent amounts of total thiamin were present in the two types of muscle, the ratio of TTP to TDP was high (5.0) in the white muscle and low (0.41) in the red muscle. ii) Rabbit anti-chicken AK1 antiserum against the purified chicken cytosolic AK1 preparation was obtained. Both AK1 activity and TTP-synthesizing activity in crude cytosol fraction of adult chicken white muscle were inhibited in parallel by the antiserum. iii) In the white muscle of normal chicken, the TTP content and AK1 activity responsible for forming either ATP or TTP were increased in a parallel manner up to day 16 after hatching, after which both remained constant. In the red muscle, on the other hand, both the TTP content and the AK1 activity were low in comparison with those in the white muscle, and were almost constant after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate: ATP phosphoryltransferase

An enzyme system catalyzing the synthesis of thiamin triphosphate consists of an enzyme (protein-bound thiamin diphosphate:ATP phosphoryltransferase), thiamin diphosphate bound to a macromolecule as substrate, ATP, Mg2+, and a low molecular weight cofactor. This system was established by combining a purified enzyme and an essentially pure, macromolecule-bound substrate prepared from rat livers. This macromolecule was found to be a protein, and the transphosphorylation of thiamin diphosphate to thiamin triphosphate with ATP and enzyme was shown to occur on this macromolecule which binds thiamin diphosphate. Free thiamin, thiamin monophosphate, thiamin diphosphate, and thiamin triphosphate have no effect on this reaction. Thus, the overall reaction is: thiamin diphosphate-protein + ATP in equilibrium thiamin triphosphate-protein + ADP. So-called thiamin diphosphate:ATP phosphoryltransferase (EC 2.7.4.15) activity was not detected in rat brain or liver. The enzyme was extracted from acetone powder of a crude mitochondrial fraction of bovine brain cortex and purified to homogeneity with a 0.6% yield after DEAE-cellulose chromatography, a first gel filtration, hydroxylapatite chromatography, chromatofocusing, and a second gel filtration. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its molecular weight was estimated to be 103,000. The pH optimum was 7.5, and the Km was determined to be 6 X 10(-4) M for ATP. ATP was found to be the most effective phosphate donor among the nucleoside triphosphates. Amino acid analysis of the purified enzyme revealed an abundance of glutaminyl, glutamyl, and aspartyl residues. Sulfhydryl reagents inhibited the enzyme reaction. Metals such as Fe2+, Zn2+, Pb2+, and Cu2+ strongly inhibited the activity. The enzyme was unstable, and glycerol (20%) and dithiothreitol (1.0 mM) were found to preserve the enzyme activity.     

Properties of the thiamin triphosphate-synthesizing activity catalyzed by adenylate kinase (isoenzyme 1)

Adenylate kinase isozyme 1 (AK1) catalyzes thiamin triphosphate (TTP) formation from thiamin diphosphate (TDP) and ADP. The properties of the TTP-synthesizing activity of purified AK1 from porcine skeletal muscle were studied. The activity was found to require TDP, ADP, and Mg2+, and ATP was only 14.4% as active as ADP. Thiamin monophosphate (TMP) and thiamin were not utilized as substrates. ADP was specific as a phosphate donor; and CDP, UDP, and GDP supported TTP formation at rates less than 1% of that with ADP. Optimal pH and temperature for the TTP-synthesizing activity were 10.0 and 37 degrees C, respectively. The activity showed saturation kinetics for both substrates, and the Km values for TDP and ADP were calculated to be 0.83 mM and 43 microM, respectively. The enzyme catalyzed the reverse reaction (TTP + AMP----TDP + ADP) and stoichiometry between TTP and TDP was demonstrated in the forward and reverse reactions.     

Identification of creatine as a cofactor of thiamin-diphosphate kinase

Thiamin-diphosphate (TDP) kinase which catalyzes thiamin triphosphate formation from TDP requires a low-molecular-mass cofactor in addition to ATP and Mg2+. The cofactor was isolated in a crystalline form from pig skeletal muscle and identified as creatine by proton NMR, mass spectrometry, infrared spectrometry and elemental analysis. The isolated cofactor and authentic creatine supported the same activity of partially purified TDP kinase at identical molar concentrations. Neither creatine phosphate nor creatinine showed activity as a cofactor. This is the first report showing evidence of the existence of a creatine-dependent enzyme.     

Metabolism of Thiamine Triphosphate in Rat Brain: Correlation with Chloride Permeability

Abstract: Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 m M ) as substrate, a 10-fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P₂ fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [β-³²P]TDP and [γ-³²P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn²⁺. These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein-bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent-solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized.     

Thiamin Deficiency in the Lamb: Changes in Thiamin Phosphate Esters in the Brain

: Concentrations of thiamin (unphosphorylated), thiamin monophosphate (TMP), thiamin diphosphate (TDP), and thiamin triphosphate (TTP) were measured in three regions of the brain of seven pairs of lambs. The lambs were maintained on a thiamin-free synthetic diet for 2, 3, or 4 weeks. Controls were pair-fed and supplemented with thiamin. The three brain regions were: (1) dorso-lateral aspect of the cortex [common site for lesions of polioencephalomalacia (PEM)]; (2) pyriform lobe of the cortex (no PEM lesions are found here); (3) white matter of the internal capsule (no PEM lesions found here). The concentration of TTP in all three sections of brain was maintained at control values for up to 4 weeks on the thiamin-deficient diet. TDP concentration decreased to 22% of control values in both regions of grey matter after 4 weeks on the diet. Unphosphorylated thiamin and TMP decreased to a smaller extent than TDP.     

Regulation of heart muscle pyruvate dehydrogenase kinase

1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [(32)P]phosphate from [gamma-(32)P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100mum) and cyclic 3':5'-nucleotides (at 10mum) had no significant effect on kinase activity. 3. The K(m) for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76mum. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The K(m) for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9-25.4mum. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The K(m) for pyruvate in the pyruvate dehydrogenase reaction was 35.5mum. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25-500mum. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10mum) the kinase activity was enhanced by low concentrations of pyruvate (25-100mum) and inhibited by a high concentration (500mum). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms (14)CO(2) from [1-(14)C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg(2+) (15mm) and by Ca(2+) (10nm-10mum) at low Mg(2+) (0.15mm) but not at high Mg(2+) (15mm).     

Mechanism of endogenous phosphorylation of microtubule proteins during GTP-induced microtubule assembly and implications for stability of the assembled structures

Cycle-purified microtubule protein from mammalian brain incorporated [32P]Pi upon incubation with [gamma-32P]GTP under the conditions used to promote assembly. This phosphorylation also occurred in the same proteins when phosphorylated with [gamma-32P]ATP and was only slightly stimulated by cAMP. GTP was a much less effective substrate than ATP. The transfer of phosphoryl groups from [gamma-32P]GTP to endogenous proteins followed a linear time-course and was stimulated by low concentrations of ATP and, more efficiently, by ADP. These data are in agreement with the predictions derived from a mechanism of phosphorylation by which [gamma-32P]GTP does not act as a phosphoryl donor for the protein kinase activity but, instead, only as a repository of high group transfer potential phosphoryl groups used to make [gamma-32P]ATP, from contaminating ADP, by means of the nucleoside diphosphate kinase activity. Using 100 mM fluoride, which suppressed protein phosphorylation without inhibiting the nucleoside diphosphate kinase activity, formation of [gamma-32P]ATP was detected. Fluoride was also able to protect microtubules from a slow depolymerization which was found to occur during long-term incubation of microtubules. This indicates that the phosphorylation observed in the presence of GTP is sufficient to destabilize microtubules.     

Identification and characterization of an ATP.Mg-dependent protein phosphatase from pig brain

Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32P-labeled myelin basic protein (MBP) and [32P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues.     

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.     

Studies on a rat-liver cell-sap protein yielding 3-[32P]-phosphohistidine after incubation with [32P]ATP and alkaline hydrolysis. Identification of the protein as ATP citrate lyase

1. The rat-liver cell-sap material from which 3-[32P]phosphohistidine was previously isolated after incubation with [gamma-32P]ATP and alkaline hydrolysis, was shown to increase about 6-fold on a high-carbohydrate diet. This increase in 32P labelling corresponded to the increase in ATP citrate lyase activity of livers of rats fed on a high-carbohydrate diet, as reported by others. 2. ATP citrate lyase [ATP:citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephopshorylating), EC 4.1.3.8] was purified from rat liver essentially according to the method of Plowman and Cleland (J. Biol. Chem., 242 (1967) 4239). The purified enzyme was incubated for a short time at 0 degree with [gamma-32P]ATP in the presence of 20 mM magnesium acetate. The phosphorylated protein was hydrolysed in alkali and the main part of the radioactivity was identified as 3-[32P]phosphohistidine. The identity of the phosphorylated amino acid was established by Dowex-1 chromatography, paper electrophoresis, paper chromatography and by analysis of the stability to acid. 3. It is concluded from these and previous results from this laboratory that ATP citrate lyase and nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) account for most of the normal rat-liver cell-sap protein which is rapidly phosphorylated by ATP.     

Identification, purification and reconstitution of thiamin metabolizing enzymes in human red blood cells.

Thiamin and its mono- (TMP), di- (TDP) and triphosphate (TTP) were assayed in adult human whole blood using high-performance liquid chromatography (HPLC). TDP and TTP were detected in red blood cells (RBC), but not in plasma. After incubation with 20 microM thiamin and 5 mM glucose for 2 h, the TDP and TTP contents of RBC increased from 111 to 222 and 0.6 to 2.2 nmol/l of packed RBC, respectively, suggesting enzymatic conversion of thiamin to TDP and then to TTP. Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) had not been isolated before from human materials, nor had cytosolic adenylate kinase (AK1, EC 2.7.4.3) in human RBC been demonstrated to catalyze the phosphorylation of TDP to TTP, although AK1 from pig and chicken skeletal muscle possess TTP-synthesizing activity. TPK and AK1 in a human RBC lysate were therefore purified by a series of the conventional techniques. The specific activity of the purified TPK, which was obtained as a single protein, was 720 nmol TDP formed/mg protein per h at 37 degrees C. A partially purified AK1 preparation catalyzed the formation of TTP from TDP (specific activity, 170 nmol/mg protein per h at 37 degrees C) in addition to its proper reaction to form ATP from ADP. After incubation of the purified TPK and AK1 with 20 microM thiamin in the presence of ATP, ADP and Mg2+ at 37 degrees C for 48 h, the amounts of TDP and TTP synthesized were 465 and 54.0 pmol/250 microliters reaction mixture, respectively. Neither TDP nor TTP was formed when TPK was omitted from the reaction mixture and an omission of AK1 resulted in the formation of TDP alone. These results indicate that thiamin is converted to TDP by TPK and, subsequently, to TTP by AK1 in human RBC.     

Is Ca2+ -Calmodulin-Dependent Protein Phosphorylation in Rat Brain Modulated by Carboxylmethylation?

Calmodulin stimulation of protein kinase activity in calmodulin-depleted preparations of rat brain cytosol or synaptosomal membranes was attenuated by prior carboxylmethylation of the enzyme source with purified protein-O-carboxylmethyltransferase. Similarly, calmodulin stimulation of highly purified Ca2+-calmodulin-dependent protein kinase was reduced if the kinase was exposed to methylating conditions prior to addition of calmodulin. Biochemical and acidic sodium dodecyl sulfate-gel electrophoretic analyses indicated that all sources of protein kinase activity were substrates for methylation. The specific activity of methyl group incorporation into protein kinase increased with increasing purity of the preparation, reaching values of 1.72 pmol CH3/micrograms protein or 0.15-1.12 mol CH3/mol of holoenzyme. Analysis of ATP binding in cytosol with the use of the photoaffinity probe [32P]8-azido-ATP indicated that carboxylmethylation reduced ATP binding. These results suggest that carboxylmethylation of Ca2+-calmodulin protein kinase may modulate the activity of this enzyme in rat brain.     

Endogenous surface phosphorylation reactions and ectokinase activity in the guinea pig T lymphocyte

The guinea pig T lymphocyte, known to interact with other cells via direct cell-to-cell contact, exhibits endogenous surface kinase activity as reflected by the appearance of four major labeled bands in autoradiographs of dried sodium dodecyl sulfate (NaDoSO4)-polyacrylamide gel electrophoresis gels when intact cells are briefly exposed to micromolar concentrations of [gamma-32P]ATP followed immediately by solubilization with NaDoSO4 to terminate the reaction. This pattern differs from the labeling of intracellular components which is seen when intact cells are incubated with 32PO4 to generate intracellular [gamma-32P]ATP when only two major labeled bands of protein with different molecular weights are seen. Of a number of modulators of lymphocyte function tested, cyclic GMP and phytohemagglutinin (PHA) caused additional bands to appear in cells exposed to [gamma-32P]ATP. The labeling of added casein was catalyzed by intact cells harvested 4 weeks after injection of animals with Freund's complete adjuvant but not earlier. These findings indicate that plasma membrane kinase activity of guinea pig T lymphocytes is accessible to the extracellular environs (ectokinase activity) and to endogenous surface substrates and that the limitation for such reactions is the availability of ATP in the extracellular component. In view of the number of circumstances under which ATP could appear outside of cells for brief periods of time, these reactions could well take place in vivo.     

ITP pyrophosphohydrolase and IDP phosphohydrolase in rat tissue.

The existence of a nucleoside triphosphate pyrophosphohydrolase specific for ITP has been demonstrated in the cytosol fraction of a variety of rat tissues. The enzyme, stable to moderate heat treatment, was present in erythrocytes as well as brain, heart, kidney, liver, lung, muscle, ovaries, spleen, testes and thymus. The specific activity of the enzyme ranges from 26 to 150 mumoles/min/g protein. In addition, evidence is given for a heat labile nucleoside diphosphate (IDP) phosphohydrolase present in most rat tissues, and particularly high in the adrenal (137 mumoles/min/g protein). An "ITP-IMP cycle" is proposed as a rgulating mechanism for intracellular levels of ATP.     

Rat Brain Synaptosomal ATP:AMP-Phosphotransferase Activity

Adenylate kinase activity (ATP:AMP-phosphotransferase; EC 2.7.4.3) was studied in various subcellular fractions of rat brain tissues. Because of the presence of other adenosine nucleotide-utilizing enzymes, adenylate kinase activity was assayed in both the forward and reverse directions by using coupled enzyme systems and by using a specific adenylate kinase inhibitor, P1,P5-di(adenosine-5') pentaphosphate. As expected, the highest specific adenylate kinase activity (2.89 mumol/min/mg of protein) was detected in the cytosolic brain fraction. However, substantial enzyme activity (0.68 mumol/min/mg) was also found in the intact synaptosomal fraction isolated on Percoll/sucrose gradients. The increased specific enzyme activity of purified synaptosomes and the differences found between the kinetic parameters of the membrane-bound and cytosolic enzyme forms suggest that the synaptosomal adenylate kinase activity cannot be attributed to the small amount of contaminating cytosol present in our preparations. The adenylate kinase enzyme adhered to purified synaptic plasma membranes and was not released by washings with isoosmotic sucrose medium. The facts that the adenylate kinase enzyme activity could be measured in intact synaptosomal preparations and that both its substrates and its inhibitors do not cross intact plasma membranes support the possibility that the synaptosomal adenylate kinase is an ecto-enzyme.     

Further observations on the properties of serum and tissue guanase from man and some animal species. Optimum pH and activation energy in the presence of 8-azaguanine.

The activation energy and the optimum pH of guanine deaminase in man, the rat, guinea pig and mouse were studied using 8-azaguanine as a substrate. The serum guanase in man and in all the animal species studied differs in activation energy from the guanase of the liver. In man, moreover, the serum guanase is also different from the brain and kidney enzyme. In the rat and guinea pig the brain enzyme has thermic activation energy different from the liver and kidney enzyme. The guanase of the serum and tissues of the guinea pig differs from the enzyme of the serum and tissues of man, rat and mouse for optimum pH.