The use of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin in the analysis of nonphosphorylated and phosphorylated rat striatal tyrosine hydroxylase at pH 7.0

The kinetics of tyrosine hydroxylase from the desalted high-speed supernatants of rat striatal homogenates were examined at pH 7.0 using different concentrations of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin, ranging from 4 μM to 1.5 mM. All analyses were performed using two different buffering solutions and their appropriate reducing systems for maintaining cofactor in the reduced state. In the presence of phosphate buffer the results show that tyrosine hydroxylase exists in two kinetically different forms with apparent K_m values for the cofactor of 16 μM (low K_m) and 2.3 mM (high K_m). Similar results were obtained using MOPS buffer. A comparative analysis of the appropriate V_max values indicates that tyrosine hydroxylase as obtained by our standard preparation procedures is predominately (95%) in the high K_m form. When the striatal supernatant was exposed to phosphorylating conditions and subsequently analyzed it appeared that the enzyme now existed totally in the low K_m form with very little change in the overall V_max. A comparison of the results using the two different buffering systems, phosphate and MOPS, revealed that following phosphorylation a large percentage of enzyme was maintained in the phosphorylated state only when using phosphate buffer. In light of the present results, we can for the first time suggest a functional significance not only for the two apparently different kinetic forms of the enzyme but also for a supporting role for phosphorylation. In vivo dopamine synthesis may be accomplished to a significant extent by the phosphorylated form of the enzyme while the non-phosphorylated form may constitute a relatively inactive reservoir which can be recruited for increased dopamine synthesis by phosphorylation.     

5′-Substituted 2′-Deoxycytidines as Non-Substrate Inhibitors of Human Deoxycytidine Kinase

Abstract

Several 5′-substituted analogues of 2′-deoxycytidine (dC), including 5′-amino2′,5′-dideoxycytidine (5′-amino-ddC), 5′-azido-2′,5′-dideoxycytidine (5′-azido-ddC) and 5′-O-ethyl-2′-deoxycytidine (5′-O-ethyl-dC), are non-substrate inhibitors of human dC kinase. Whereas 5′-amino-ddC inhibits phosphorylation of deoxyadenosine (dA) with K_i ～ 1 μ inhibition of phosphorylation of dC is bimodal and more effective at low inhibitor concentrations. In particular 5′-O-ethyl-dC inhibits phosphorylation of dA 10-fold more effectively (K_i ～ 3 μ) than that of dC (K_i ～ 30 μ). For 5′-azido-ddC inhibition was shown directly to be competitive with respect to substrate.     

Substrate/Inhibitor Properties of Human Deoxycytidine Kinase (dCK) and Thymidine Kinases (Tk1 And Tk2) Towards the Sugar Moiety of Nucleosides,Including O′-Alkyl Analogues

Abstract

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono-and di-O′-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower K_i values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3′-hexanoylamino-2′,3′-dideoxythymidine, with a K_i of ～600 μM for TK1 and ～0.1 μM for TK2. 3′-OMe-dC was a superior inhibitor of dCK to its 5′-O-methyl congener, consistent with possible participation of the oxygen of the (3′)-OH or (3′)-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly α-dT was a good substrate of both TK1 and TK2, with K_i values of 120 and 30 μM for TK1 and TK2, respectively; and a 3′-branched α-L-deoxycytidine analogue proved to be as good a substrate as its α-D-counterpart. Several 5 ′-substituted analogues of dC were     

Enzymic synthesis of lignin precursors. Purification of properties of the S-adenosyl-l-methionine: caffeic acid 3-O- methyltransferase from soybean cell suspension cultures.

A 3-O-methyltransferase which catalyzes the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified about 60-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The enzyme utilized, in addition to caffeic acid (K_m = 133 μM), 5-hydroxyferulic acid (K_m = 55 μM), 3,4,5-trihydroxy-cinnamic acid (K_m = 100 μM), and protocatechualdehyde (K_m = 50 μM) as substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of ferulic acid, of ortho-, meta-, and para-coumaric acids, and of the flavonoid compounds quercetin and luteolin. The methylation of caffeic acid and 5-hydroxyferulic acid showed a pH optimum at 6.5–7.0. No stimulation of the reaction velocity was observed when Mg²⁺ ions were added. EDTA did not inhibit the reaction. The K_m for S-adencsyl-l-methionine was 15 μm. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine (K_i = 6.9 μM).     

Choline sulfokinase of Penicillium chrysogenum: partial purification and kinetic mechanism.

Choline sulfokinase (3′-phosphoadenosine 5′-phosphosulfate (PAPS):choline sulfotransferase, EC 2.8.2.6) was purified approximately 30-fold from the mycelium of Penicillium chrysogenum. The K_m for PAPS is 12 μm. The enzyme is remarkably specific for the adenosine 3′,5′ (or 2′-5′)-diphosphate moiety. 3′,5′-ADP (PAP) has a K_i of 2.5 to 14 μm (depending on the choline concentration) whereas the K_i values of 3′-AMP, 5′-AMP, and 5′-ADP are at least 300-fold higher. The enzyme is also highly specific for choline (K_m = 17 μM). Of a number of other amino alcohols tested, none were potent inhibitors and only dimethylaminoethanol served as a reasonably good substrate (K_m = 800 μmV = 35% of V with choline). Triethylaminoethanol was a significantly poorer substrate (K_m = 2800 μM; V = 2% of V with choline). The purified enzyme is relatively stable when stored frozen in the presence of 25% sucrose. In the absence of sucrose, the maximum activity decreases and the K_m for choline increases. (The K_m for PAPS remains constant.) The age-inactivated enzyme can be restored to full activity (original V and K_m for choline) by a 10-min preincubation with 50 mm mercaptoethanol. However, prolonged incubation (24 h) with 50 mm mercaptoethanol results in irreversible denaturation. Initial velocity studies established that the enzyme follows a sequential kinetic mechanism. Product inhibition studies suggest a rapid equilibrium random binding sequence. Choline-O-phosphate (a dead-end inhibitor) is linearly competitive with choline and a linear mixed type inhibitor with respect to PAPS. Choline analogs lacking the alcohol (or ester) group (e.g., trimethylammonium, neurine, chlorocholine) are competitive dead-end inhibitors with respect to choline but are uncompetitive with respect to PAPS. Thiocholine is a linear mixed type inhibitor with respect to PAPS, but the reciprocal plots are almost parallel. These results suggest that the analogs lacking an oxygen atom have a negligible affinity for the free enzyme and bind predominantly to the enzyme-PAPS complex.     

Synthesis of carbocyclic pyrimidine nucleosides and their inhibitory activities against Plasmodium falciparum thymidylate kinase

Plasmodium falciparum thymidylate kinase (PfTMK) is a promising antimalarial target due to its unique substrate specificity. Recently, we reported that 2′,3′-dideoxycarbocyclic thymidine showed moderate inhibitory activity and reported the related structure–activity relationship for inhibitors against PfTMK. In this study, we have designed and synthesized enantioselective 2′,3′-dideoxycarbocyclic pyrimidine nucleosides based on our previous results and screened them for inhibitory activity against PfTMK. The most potent inhibitor showed K_i^TMP and K_i^dGMP values of 14 and 20 μM, respectively. The fluorinated dideoxy derivative (-)-7, exhibited lower K_i^TMP and higher K_i^dGMP compared with that of the parent compound (K_i^TMP, K_i^dGMP equals 20 and 7 μM, respectively). The modification of carbocyclic pyrimidine nucleosides is a promising strategy for developing powerful PfTMK inhibitors.     

High tyrosine-specific protein kinase activity in normal human peripheral blood lymphocytes

Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60^src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent K_m values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg²⁺ to Mn²⁺ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N⁶;O^2′-dibutyryl cyclic AMP (100 μM), N⁶;O^2′-dibutyryl cyclic GMP (100 μM), Ca²⁺ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with M_r 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase.     

Evaluation of α-hydroxycinnamic acids as pyruvate carboxylase inhibitors

Through a structure-based drug design project (SBDD), potent small molecule inhibitors of pyruvate carboxylase (PC) have been discovered. A series of α-keto acids (7) and α-hydroxycinnamic acids (8) were prepared and evaluated for inhibition of PC in two assays. The two most potent inhibitors were 3,3′-(1,4-phenylene)bis[2-hydroxy-2-propenoic acid] (8u) and 2-hydroxy-3-(quinoline-2-yl)propenoic acid (8v) with IC₅₀ values of 3.0 ± 1.0 μM and 4.3 ± 1.5 μM respectively. Compound 8v is a competitive inhibitor with respect to pyruvate (K_i = 0.74 μM) and a mixed-type inhibitor with respect to ATP, indicating that it targets the unique carboxyltransferase (CT) domain of PC. Furthermore, compound 8v does not significantly inhibit human carbonic anhydrase II, matrix metalloproteinase-2, malate dehydrogenase or lactate dehydrogenase.     

Glucose transport and metabolism in Gymnodinium breve

Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K_FD of 11 μM and a V_max of 1 × 10^?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (K_i = 40 μM), mannose (K_i = 12O μM), and 2-deoxy-d-glucose (K_i = 190 μM) and non-competitively inhibited by galactose (K_i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.     

Inhibition of dopamine β-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-car☐ylic acid (QCA), 1-isoquinolinecar☐ylic acid (IQCA), 2,2′-bi-1H-imidazole (2,2′-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine β-hydroxylase with respect to ascorbate (K_is = 5.7(±0.9) μM, 0.14(±0.03) mM, 0.80(±0.20) mM; n_H= 1.4(±0.1), 1.8(±0.4), 2.8(±0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine β-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (K_is = 1.9(±0.3) μM, 21(±6) μM, and 0.9(±0.3) μM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine β-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine β-hydroxylase.     

Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which l-dopa is formed from the substrate l-tyrosine. l-Dopa concentration and activity of l-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum l-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH₄, and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K _m value of 808.63 μM for l-tyrosine at 37°C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 μM l-tyrosine. Higher concentrations of the cofactor 6-MPH₄, however, completely inhibited the synthesis of l-dopa. Tyrosine hydroxylase converted specific monophenols such as l-tyrosine (808.63 μM) and tyramine (K _m 1.1 mM) to diphenols l-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

3′-Oxo-, amino-, thio- and sulfone-acetic acid modified thymidines: Effect of increased acidity on ribonuclease A inhibition

A family of 3′-functionalized thymidines carrying XCH₂COOH (X = O, NH, S, SO₂) groups has been designed as inhibitors of RNase A. This is because it is possible to manipulate the overall acidity of this new class of nucleic ‘acids’ by changing X from oxygen to the SO₂ group in the series. It is also expected that the acyclic nature of the XCH₂COOH group would provide enough flexibility to the –COOH group to have maximum interactions with the catalytic subsite P₁ of RNase A. As the –SO₂CH₂COOH substituted derivative showed better potency partially because of the increased acidity of the –COOH group, the inhibitory properties of both 5′-substituted and 3′,5′-disubstituted sulfone acetic acid modified thymidines were investigated. Two –SO₂CH₂COOH groups were incorporated with the expectation of targeting two phosphate binding sites simultaneously. Thus, 3′,5′-dideoxy-3′,5′-bis-S-[(carboxymethyl)sulfonyl]thymidine emerged as the best inhibitor in this series with a K_i value of 25 ± 2 μM.     

Binding of pyridine coenzymes to the β-subunit of the voltage sensitive potassium channels

The β-subunit of the voltage-sensitive K⁺ channels shares 15–30% amino acid identity with the sequences of aldo–keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of K_vβ2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The K_D values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1±0.007 and 0.05±0.006 M, respectively — indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the α-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the K_D for NADH and NAD were between 1–3 μM whereas the K_D for FAD was >10 μM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2′ phosphate at the binding site. The selectivity of K_vβ2.1 for NADPH over NADP may be significant in regulating the K⁺ channels as a function of the cellular redox state.     

Dopamine biosynthesis from L-tyrosine and L-phenylalanine in rat brain synaptosomes: preferential use of newly accumulated precursors.

Abstract— The biosynthesis of dopamine (DA) from L-tyrosine (Tyr) and L-phenylalanine (Phe) was investigated using synaptosomes prepared from the striatum and olfactory tubercle of the rat. The formation of ¹⁴CO₂ from either carboxyl labeled precursor occurred exclusively within the synaptosome following hydroxylation and subsequent decarboxylation. The optimum pH for the formation of DA was 6.2 and was independent of precursor and tissue source. As pH increased beyond this optimum, synthesis from Tyr declined more rapidly than that from Phe. Synthesis obeyed Michaelis–Menton kinetics when expressed as a function of the specific activity of precursor in the medium. It was characterized by an overall K_m (approx 0.9 μM) which was independent of precursor and tissue source, and was considerably lower than the K_t for accumulation of precursor by synaptosomes (15.3 and 13.3 μM for Tyr and Phe, respectively). While each precursor was an uncompetitive inhibitor of DA synthesis from the opposing labeled amino acid, Tyr was a more effective inhibitor of synthesis from Phe (K_i= 1.5 μM) than was Phe an inhibitor of synthesis from Tyr (K_i= 9.2 μM). Tryptophan inhibited synthesis competitively (K_i= 15.2 and 13.2 μM for synthesis from Tyr and Phe, respectively), and DA inhibited non-competitively (K_i= 1.1 and 0.42 μM for Tyr and Phe, respectively). A model of DA synthesis within the synaptosome is presented which attempts to integrate these data. A major feature of this schema is the proposal that newly accumulated precursor does not mix rapidly with endogenous precursor pools but rather is preferentially converted to DA.     

Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential

Triterpenoids are in the focus of scientific interest, and they were evaluated for many pharmacological applications among them their ability to act as inhibitors of cholinesterases. These inhibitors are still of interest as drugs that improve the life quality of patients suffering from age-related dementia illnesses especially of Alzheimer’s disease. Herein, we prepared several derivatives of ursolic and oleanolic acid and screened them in Ellman’s assays for their ability to inhibit acetylcholinesterase and/or butyrylcholinesterase, and for each of the active compounds the type of inhibition was determined. As a result, several compounds were shown as good inhibitors for acetylcholinesterase and butyrylcholinesterase even in a micromolar range. An ursolic acid derived hydroxyl-propinyl derivative 10 was a competitive inhibitor for butyrylcholinesterase with an inhibition constant of K_i = 4.29 μM, and therefore being twice as active as gold standard galantamine hydrobromide. The best inhibitor for acetylcholinesterase, however, was 2-methyl-3-oxo-methyl-ursoloate (18), acting as a mixed-type inhibitor showing K_i = 1.72 µM and K_i′ = 1.28 μM, respectively.     

Effect of Dopamine on Activation of Rat Striatal Adenylate Cyclase by Free Mg2+ and Guanyl Nucleotides1

Abstract: Stimulation of rat striatal adenylate cyclase by guanyl nucleotides was examined utilizing either MgATP or magnesium 5′-adenylylimidodiphos-phate (MgApp(NH) p) as substrate. GTP and 5′- guanylylimidodiphosphate (Gpp(NH) p) stimulate adenylate cyclase under conditions where the guanyl nucleotide is not degraded. The apparent stimulation of adenylate cyclase by GDP is due to an ATP-dependent transphosphorylase present in the tissue which converts GDP to GTP. We conclude that GTP is the physiological guanyl nucleotide responsible for stimulation of striatal adenylate cyclase. Dopamine lowers the K_a for Gpp(NH) p stimulation twofold, from 2.4 μM to 1.2 μM and increases maximal velocity 60%. The kinetics of Gpp(NH) p stimulation indicate no homotropic interactions between Gpp(NH) p sites and are consistent with one nonessential Gpp(NH) p activator site per catalytic site. Double reciprocal plots of the activation by free Mg²⁺ were concave downward, indicating either two sets of sites with different affinities or negative cooperativity (Hill coefficient = 0.3, K_0.5= 23 mM). The data conform well to a model for two sets of independent sites and dopamine lowers the K_a for free Mg²⁺ at the high-affinity site threefold, from 0.21 mM to 0.07 mM. The antipsy-chotic drug fluphenazine blocks this shift in K_a due to dopamine. Dopamine does not appreciably affect the affinity of adenylate cyclase for the substrate, MgApp(NH) p. Therefore, dopamine stimulates striatal adenylate cyclase by increasing the affinity for free Mg²⁺ and guanyl nucleotide and by increasing maximal velocity.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Effects of thyrotropin,prostaglandin E1 and iodide on cyclic 3′,5′-AMP concentration in dog thyroid slices

The kinetics and concentration effect on the relationship of thyrotropin (TSH) action on cyclic 3′,5′-AMP concentration has been studied in dog thyroid slices in vitro. TSH markedly increased cyclic 3′,5′-AMP level after 5 min, the effect reached a plateau after 10–60 min and slowly declined afterwards. TSH enhanced in parallel the cyclic 3′,5′-AMP level and the binding of iodide to proteins. For this latter effect of TSH, the four criteria of the validity of the Sutherland model for a hormonal action are therefore fulfilled. The effect of TSH on cyclic 3′,5′-AMP concentration in thyroid did not require the presence of a methylxanthine inhibitor of cyclic 3′,5′-AMP phosphodiesterase in the medium. Prostaglandin E1 increased cyclic 3′,5′-AMP levels in control and stimulated slices. The omission of Ca²⁺ in the incubation medium decreased the action of TSH but partial replacement of Na⁺ by K⁺ had little effect. Iodide, 1 μM to 100 μM, inhibited the action of TSH. This inhibitory effect was relieved by NaClO₄, methimazole and propylthiouracil (1 mM). The possible role of this inhibitory effect in an intracellular regulatory mechanism is discussed.