Retinal Hexokinase: Kinetic Properties and the Effect of Cyclic 3',5'-Adenosine Monophosphate

Abstract: We measured hexokinase (EC 2.7.1.1) activity in particulate and soluble fractions isolated from bullfrog ( Rana catesbeiana ) retinas. Seventy-three percent of the hexokinase (HK) activity was associated with the particulate fraction, 27% with the soluble fraction. Both HK fractions could phosphorylate fructose, glucose, 2-deoxy-d-glucose, and mannose, but not galactose. The K _m for glucose was 0.14 m M , for 2-deoxy-d-glucose. 3.6 m M . With glucose as substrate, the V _max for particulate HK was 125–148 μ M retina⁻¹ min⁻¹, for soluble HK, 37 μ M retina⁻¹ min⁻¹. Product inhibition of particulate HK activity by glucose 6-phosphate was marked, whereas 2-deoxy-d-glucose 6-phosphate did not inhibit the activity. Cyclic AMP stimulated the HK activity of both retinal fractions nearly twofold at concentrations of 0.2–0.8 m M; AMP was much less effective in this regard.     

CYCLIC NUCLEOTIDE METABOLISM IN ELECTROPLAX OF ELECTROPHORUS ELECTRICUS

Abstract— In order to describe the regulation of cyclic nucleotide metabolism in a cholinergic tissue, the properties of cyclic nucleotide phosphodiesterase were determined in electroplax of Electrophorus electricus and compared to those of mammalian brain. Electroplax phosphodiesterase was Mg²⁺ -dependent. localized in the soluble fraction and displayed normal linear Lineweaver-Burk kinetics ( K _m: cyclic AMP. 1.4 μ m ; cyclic GMP, 0.54 μ m ). No low affinity (i.e. high K _m) activity was detected. These results were correlated with comparatively low tissue levels of cyclic AMP (67 pmol/g) and cyclic GMP (3.2 pmol/g). Attempts were made to detect calcium-dependent phosphodiesterase because of the presence of large amounts of the calcium-dependent regulator protein (CDR) in electroplax, as this protein has been shown to activate brain phosphodiesterase. Assay with EGTA under a variety of conditions revealed that no calcium-dependent activity could be detected. Preparation of CDR-deficient phosphodiesterase also failed to produce calcium-dependent activity. Assay of phosphodiesterase in other cholinergic tissues revealed calcium-dependent activity in Electrophorus muscle and rat diaphragm but not in Torpedo electroplax. The results suggest that calcium-dependent activity is not a significant portion of phosphodiesterase in electroplax and indicate alternate roles for CDR in electric tissue.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF THE BOVINE RETINA: ACTIVITY, SUBCELLULAR DISTRIBUTION AND KINETIC PARAMETERS

Abstract— High phosphodiesterase activity for cyclic AMP and cyclic GMP was found in subcellular fractions of the bovine retina with more rapid hydrolysis of cyclic GMP than cyclic AMP in each fraction. Rod outer segments (ROS) and the supernatant fraction had highest activity. High enzyme activity remained associated with ROS membranes through several steps of purification by gradient centrifugation. A complex kinetic pattern was observed for cyclic AMP hydrolysis by the supernatant fraction yielding two values for K _m; a simple kinetic pattern was observed with cyclic GMP hydrolysis in supernatant and for both cyclic nucleotides in preparations of purified outer segments. Phosphodiesterase activity of outer segments was enhanced by Mg²⁺. Mn²⁺ and inhibited by EDTA. Cyclic AMP had relatively little effect on the hydrolysis of cyclic GMP in supernatant or ROS while cyclic GMP inhibited hydrolysis of cyclic AMP in both fractions.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF HUMAN CEREBROSPINAL FLUID

Abstract— Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'–GMP (cGMP) phosphodiesterase activities were found in human cerebrospinal fluid (CSF) using low substrate concentration (0.4μM). More rapid hydrolysis of cGMP than that of cAMP was observed in human CSF. However, cGMP hydrolytic activity of CSF was very much lower (0.3 pmol/min/ml CSF) than that of human cerebral cortex (33.7 nmol/min/g wet cortex). The pH optimum was found to be 8.0 (cGMP phosphodiesterase) and 7.5 (cAMP phosphodiesterase). The maximum stimulation of both cAMP and cGMP phosphodiesterase was achieved at 4 mM-MgCl₂. Cyclic AMP had relatively little effect on the hydrolysis of cGMP in CSF and the cortex, while cGMP inhibited hydrolysis of cAMP in both tissues. Snake venom was found to stimulate cAMP and cGMP phosphodiesterase activity of CSF, by 60% and 110% respectively. This stimulation by snake venom was also observed in the cortex phosphodiesterase, but was not observed in human plasma or thyroid phosphodiesterase. When CSF was applied to Sepharose 6B column, cGMP phosphodiesterase was separated into three different molecular forms. A plot of activity against substrate concentration using peak I (largest molecular size) revealed a high affinity ( K _m= 2.6μM) and a low affinity ( K _m= 100μM) for cAMP suggesting the existence of at least two molecular forms of the enzyme. On the other hand, using a cGMP as substrate the only one K _m value (1.90 μm) was obtained. These K _m values of CSF enzymes described above were close to those obtained from human cerebral cortex preparations. The enzyme under peak I corresponded to the cortex enzyme when judged from its molecular size and stimulation by snake venom. It seems likely from our results that at least a part of CSF phosphodiesterase originates from the central nervous system.     

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower K _ms for both tyrosine (7 μ M ) and reduced pterin cofactor (110 μ M ) relative to the soluble enzyme (47 μ M and 940 μ M , respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the K _m of the enzyme for tyrosine to 27 μ M and the V _max by 60% without changing the K _m for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A₂ increased the K _m for tyrosine to 48 μ M increased the V _max and increased the K _m for cofactor to 560 μ M . The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A₂ treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.     

MODULATION OF CYCLIC AMP LEVELS IN THE BOVINE SUPERIOR CERVICAL GANGLION BY PROSTAGLANDIN E, AND DOPAMINE

Abstract— Dopamine, norepinephrine, carbamylcholine and PGE₁ (prostaglandin E₁). increased cyclic AMP concentrations in slices of bovine superior cervical ganglia. PGF_1α was less effective and neither PGE₂ nor PGF_2α had any effect. Dopamine and PGE, alone or in combination, did not modify low K _m cyclic AMP phosphodiesterase activity. Combinations of dopamine and PGE, showed a marked synergistic effect, increasing ganglionic cyclic AMP to a much greater extent than that observed when the two compounds were tested alone. Norepinephrine (10 μ M) , which increased cyclic AMP as much as 10 μ m -dopamine, showed no synergistic effect when tested in the presence of PGE₁ or other PGs. Phentolamine, fluphenazine and triflupromazine blocked the dopamine effect without suppressing its synergism with PGE₁ Adenylate cyclase of synaptosomes isolated from the ganglia under a variety of experimental conditions appeared to be as responsive to PGE₁ as the slices, but it was poorly stimulated by dopamine and was not synergistically modulated by dopamine in the presence of PGE₁
These and other data are interpreted as indicating the presence of both a PGE₁-sensitive and a PGE₁-modulated dopamine-sensitive adenylate cyclase in the cervical ganglion. These adenylate cyclases are tentatively assigned to pre- and post-synaptic structures respectively.     

Biochemistry of an Olfactory Purinergic System: Dephosphorylation of Excitatory Nucleotides and Uptake of Adenosine

Abstract: The olfactory organ of the spiny lobster, Panu-lirus argus , is composed of chemosensory sensilla containing the dendrites of primary chemosensory neurons. Receptors on these dendrites are activated by the nucleotides AMP, ADP, and ATP but not by the nucleoside adenosine. It is shown here that the lobster chemosensory sensilla contain enzymes that dephosphorylate excitatory nucleotides and an uptake system that internalizes the nonexcitatory dephosphorylated product adenosine. The uptake of [³H]-adenosine is saturable with increasing concentration, linear with time for up to 3h, sodium dependent, insensitive to moderate pH changes and has a K _m of 7.1 μ M and a V_max of 5.2 fmol/sensillum/min (573 fmol/μg of protein/min). Double-label experiments show that sensilla dephosphorylate nucleotides extracellularly; ³H from adenine-labeled AMP or ATP is internalized, whereas ³²P from phosphate-labeled nucleotides is not. The dephosphorylation of AMP is very rapid; ³H from AMP is internalized at the same rate as ³H from adenosine. Sensillar 5'-ectonucleotidase activity is inhibited by ADP and the ADP analog α,β-methylene ADP. Collectively, these results indicate that the enizymes and the uptake system whereby chemosensory sensilla of the lobster inactivate excitatory nucleotides and clear adenosine from extracellular spaces are very similar to those present in the internal tissues of vertebrates, where nucleotides have many neuroactive effects.     

[3H]GLYCOGEN HYDROLYSIS IN BRAIN SLICES: RESPONSES TO NEUROTRANSMITTERS AND MODULATION OF NORADRENALINE RECEPTORS

Abstract— Different agents have been investigated for their effects on [C³H]glycogen synthesized in mouse cortical slices. Of these noradrenaline, serotonin and histamine induced clear concentration-dependent glycogenolysis.
[C³H]Glycogen hydrolysis induced by noradrenaline appears to be mediated by beta-adrenergic receptors because it is completely prevented by timolol, while phentolamine is ineffective. It seems to involve cyclic AMP because it is potentiated in the presence of isobutylmethylxanthine; in addition dibutyryl cyclic AMP (but not dibutyryl cyclic GMP) promotes glycogenolysis.
Lower concentrations of noradrenaline were necessary for [C³H]glycogen hydrolysis (EC₅₀= 0.5μM) than for stimulation of cyclic AMP accumulation (EC₅₀= 8μM).
After subchronic reserpine treatment the concentration-response curve to noradrenaline was significantly shifted to the left (EC₅₀= 0.09 ± 0.02 μM as compared with 0.49 ± 0.08 μM in saline-pretreated mice) without modifications of either the basal [C³H]glycogen level, maximal glycogenolytic effect, or the dibutyryl cAMP-induced glycogenolytic response.
In addition to noradrenaline, clear concentration-dependent [³H]glycogen hydrolysis was observed in the presence of histamine or serotonin. In contrast to the partial [³H]glycogen hydrolysis elicited by these biogenic amines, depolarization of the slices by 50 mM K⁺ provoked a nearly total [C³H)glycogen hydrolysis.     

Cyclic nucleotide phosphodiesterases from rat anterior pituitary. Characterization of multiple forms and regulation by protein activator and Ca+.

Phosphodiesterase activities for adenosine and guanosine 3':5'-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphadiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N'-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 10500 X g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 muM substrate being 20 muM and 24 muM for cyclic AMP and 7 muM and 10 muM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6--7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.     

Characterization of Serotonin N-Acetyltransferase in the Lateral Eye of the Green Frog Rana perezi: Protective Action of EGTA

Abstract: The kinetics of seRotonin N -acetyltransferase (NAT) from the lateral eye of Rana perezi have been characterized. NAT from ocular tissue reached maximal activity at a phosphate buffer concentration of 250 m M and a pH of 6.5. Reaction linearity was highly conserved within the homogenate fraction range tested (0.033-0.33). The time course of ocular NAT reaction showed a high linearity at 25 and 35°C. K _m and V_max estimations for acetyl-CoA at a 10 m M tryptamine concentration were 63.3 μ M and 4.42 nmol/h per eye, respectively. Regardless of the acceptor amine (tryptamine or serotonin), the K _m was not affected by the acetyl-CoA concentration (50 or 250 μ M ), whereas the V _max was significantly increased at a 250 μ M acetyl-CoA concentration. Ocular NAT showed a higher affinity for serotonin ( K _m= 20.7 μ M ) than for tryptamine ( K _m= 48-60 μ M ); V _max however, was similar for both substrates. Acetyl-CoA does not protect ocular NAT; in contrast, the use of EGTA (4 m M ) in the assay is essential to protect the enzyme because NAT in ocular crude homogenate shows rapid inactivation. This result suggests that intracellular calcium levels are involved in the NAT inactivation mechanisms in frog ocular tissue.     

Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1)

Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent Km values of 125 and 30 microM, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP anc cyclic GMP; the apparent Km values for these substrates were similar (90 and 100 microM, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 microM. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs.     

Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus . Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C₄−C₁₀) tested. Enzyme A appeared to be a tetramer ( M _r 70 000) with identical subunits ( M _r 44 000) and enzyme B had a similar M _r of 168 000 (containing M _r 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. K _m values of 44 μM and 394 μM for acetoacetyl-CoA, and 16 μM and 93 μM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave K _m values of 1.1 mM and 230 μM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases.     

Soils contain two different activities for oxidation of hydrogen

Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H₂ mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K _m values for H₂, one at about 10–40 nM dissolved H₂, the other at about 1.2–1.4 μM H₂. The low- K _m activity was less sensitive to chloroform fumigation than the high- K _m activity. If sterile soil was amended with Paracoccus denitrificans or a H₂-oxidizing strain isolated from compost soil, it exhibited only a high- K _m (0.7–0.9 μM) activity. It also failed to utilize H₂ mixing ratios below a threshold of 1.6–3.0 ppmv H₂ (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H₂ oxidation was determined from the decrease of dissolved H₂. However, H₂ was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H₂: (1) a high- K _m, high-threshold activity which apparently is due to aerobic H₂-oxidizing bacteria, and (2) a low- K _m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.     

Aldehyde Dehydrogenases in Rat Brain. Subcellular Distribution and Properties

Abstract: Kinetic studies suggested the presence of several forms of NAD-dependent aldehyde dehydrogenase (ALDH) in rat brain. A subcellular distribution study showed that low- and high- K _m activities with acetaldehyde as well as the substrate-specific enzyme succinate semialdehyde dehydrogenase were located mainly in the mitochondrial compartment. The low- K _m activity was also present in the cytosol (<20%). The low- K _m activity in the homogenate was only 10–15% of the total activity with acetaldehyde as the substrate. Two K _m values were obtained with both acetaldehyde (0.2 and 2000 μ m ) and 3,4-dihydroxyphenylacetaldehyde (DOPAL) (0.3 and 31 μ m ), and one K _m value with succinate semialdehyde (5 μ m ). The main part of the aldehyde dehydrogenase activities with acetaldehyde, DOPAL, and succinate semialdehyde, but only little activity of the marker enzyme for the outer membrane (monoamine oxidase, MAO), was released from a purified mitochondrial fraction subjected to sonication. Only small amounts of the ALDH activities were released from mitochondria subjected to swelling in a hypotonic buffer, whereas the main part of the marker enzyme for the intermembrane space (adenylate kinase) was released. These results indicate that the ALDH activities with acetaldehyde, DOPAL and succinate semialdehyde are located in the matrix compartment. The low- K _m activity with acetaldehyde and DOPAL, but not the high- K _m activities and succinate semialdehyde dehydrogenase, was markedly stimulated by Mg²⁺ and Ca²⁺ in phosphate buffer. The low- and high- K _m activities with acetaldehyde showed different pH optima in pyrophosphate buffer.     

Heterogeneity of β-Adrenoceptors in Guinea-Pig Brain: Radioligand Binding and Cyclic Nucleotide Generation

Abstract: In this report, we have examined the radioligand binding and second messenger signalling characteristics of β-adrenoceptors in the guinea-pig brain. [¹²⁵I]lodocyanopindolol ([¹²⁵I]ICYP)-labelled sites in the cerebellum and cerebral cortex were of similar densities ( B _max 34 and 24 fmol·mg⁻¹) and affinities ( K _D 20 and 55 p M ), respectively. Analysis of competition for [¹²⁵I]ICYP binding in the cerebellum was compatible with the presence of a β₂-adrenoceptor. In this tissue, isoprenaline evoked a cyclic AMP stimulation, and also potentiated cyclic GMP accumulations evoked in the presence of a nitric oxide donor, consistent with mediation via a β₂-adrenoceptor. The [¹²⁵I]ICYP binding profile in the cerebral cortex did not comply with those previously described for β-adrenoceptor subtypes, and isoprenaline failed to alter significantly cyclic AMP accumulation in the cerebral cortex, hippocampus, or neostriatum, even in the presence of forskolin or a phosphodiesterase inhibitor. Isoprenaline was also without effect on cyclic GMP accumulation or phosphoinositide turnover in the cerebral cortex. These results suggest that the guinea-pig cerebellum expresses a functional β₂-adrenoceptor coupled to cyclic AMP generation, and potentiation of cyclic GMP accumulation. However, the guinea-pig cerebral cortex displays binding sites that exhibit β-adrenoceptor-like pharmacology but fail to show functional coupling to cyclic AMP, cyclic GMP, or phosphoinositide signalling systems.     

Binding of cyclic AMP and cyclic GMP to renal cortical homogenates. Relationship with phosphorylation

This study examined the binding of both cyclic AMP and cyclic GMP to receptor proteins in particulate and soluble subfractions of renal cortical homogenates from the golden hamster. The binding of both nucleotides was compared to subsequent effects of both nucleotides on the phosphorylation of histone from identical fractions. Cyclic AMP binding and cyclic AMP-dependent protein kinase activity predominated in the cytosol, with some binding and enzyme activity also detected in particulate fractions. Cyclic GMP and cyclic GMP-dependent protein kinase activity could only be demonstrated in cytosolic fractions and represented only 20-30% of cyclic AMP-dependent activity in this fraction. Binding of both nucleotides was highly specific, however, cyclic AMP showed some interaction with cyclic GMP binding. Evidence suggesting that each nucleotide interacts with a specific protein kinase was as follows: both the binding activity of the cyclic nucleotides and their combined protein kinase activity show additivity; cyclic AMP and cyclic GMP binding activity could be separated on sucrose gradients; cyclic AMP and cyclic GMP protein kinase activity could be separated with Sephadex G-100 chromatography, after preincubation of homogenate supernatants with either cyclic AMP or cyclic GMP. The results demonstrate the presence of both cyclic AMP- and cyclic GMP-dependent protein kinase in renal cortex.     

N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.     

Kinetic Properties of Bovine Pineal Tryptophan-5-Monooxygenase Activated by an Endogenous Activating Substance

Abstract: We previously reported that an endogenous activating substance different from bovine serum albumin, phospholipids and heparin, exists in the extract from bovine pineal glands and that this substance interacts with tryptophan-5-monooxygenase under reducing conditions with sulfhydryl reagents, to stimulate monooxygenase activity. The present paper reports that the activating substance is of peptide nature; that it is sensitive to trypsin-digestion; and that it does not change the apparent K _m's for substrates, L-tryptophan and oxygen, and coenzyme, reduced biopterin or DMPH₄: but that it increases the V _max 1.5- to 2.3-fold. These results suggest that an activating protein, present in some particles of the cell structure, activates tryptophan-5-monooxygenase under the regulation of a sulfhydryl compound. The apparent K _m's for reduced biopterin and DMPH₄ were 77.2μM and 294 μM, respectively. The apparent K _m's for L-tryptophan and oxygen with reduced biopterin were 15.0 μM and 4.7%, respectively: with DMPH₄, they were 11.0 μM and 8.5%, respectively. Significant inhibition of both L-tryptophan and oxygen was observed with reduced biopterin, but not with DMPH₄ (at the tested concentrations of up to 0.5 MM and 20%, respectively).     

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity ( K _m, 2.6 μM) and, although it also binds betaine ( K _m, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX ( K _m, 24 μM) and the betaine-specific SBP BetX ( K _m, 0.6 μM). Unlike most ABC transporter loci, caiX , betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.