Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP

Spinach chloroplasts were able to photophosphorylate the ADP analog α,β-methylene adenosine 5′-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and ³²P_i, the ³²P label was incorporated into α,β-methylene adenosine 5′-triphosphate (AOPCPOP).In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-P_i exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9.Photophosphorylation of AOPCP was inhibited by the α,β- and β,γ-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-P_i exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-P_i exchange was inhibited by the β,γ-methylene analog of ATP.Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Inhibition of the peptidyltransferase acceptor site by 2′(3′)-O-cycloleucyl- and α-aminoisobutyryl derivatives of cytidylyl-(3′–5′)adenosine

2′(3′)-O-(N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5′-O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2′- and 3′-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2′,5′-di-O-tetrahydropyranylcytidine 3′-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3′→5′)2′(3′)-O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3′→5′)2′(3′)-O-(α-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[¹⁴C]Phe-tRNA·poly(U)·70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10^?5 M puromycin) was achieved at 9.5·10^?6 M of compound 1c and 9·10^?5 M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[¹⁴C]Phe to the A site of peptidyltransferase in a system containing tRNA^Phe·poly(U)·70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of α-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the α-substituent.     

2,4,6-Trinitrobenzenesulfonate labels an essential amino group near the bound phosphate at the catalytic site of mitochondrial F1-ATPase

The amino reagent 2,4,6-trinitrobenzenesulfonate (TNBS) was found to inactivate mitochondrial F₁-ATPase through covalent labeling, which was not reversed by dithiothreitol. The observed rate of inactivation was retarded by inorganic phosphate, but enhanced by prior labeling of F₁ with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1). These observations are consistent with the presence of an essential amino group near the bound inorganic phosphate at the catalytic site of F₁. A comparison of the observed protection of F₁ from NBD-C1 and 5′-p-fluorosulfonyl-benzoyladenosine (FSBA) respectively by inorganic phosphate and by 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP) suggests that NBD-C1 labels an essential Tyr residue in the positively charged locus for binding the polyphosphate end of ATP, and that FSBA labels an essential Tyr residue in the more hydrophobic locus for binding the adenosine moiety of ATP at the catalytic site of F₁.     

Dynamic affinity chromatography of heavy meromyosin subfragment-1

Heavy meromyosin subfragment-1 and its trinitrophenylated derivative 3ave been chromatographed on immobilized ATP, ADP and adenosine 5′-(β,γ-imino)triphosphate affinity chromatography columns, in the presence and in the absence of Mg²⁺ or Ca²⁺. Splitting of bound ATP was followed by using [γ-^{3 2}P]ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5′(β,γ-imino)triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the P_i-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.     

Adenosine 5'-O(3-thiotriphosphate) in the control of phosphorylase activity

Rabbit muscle phosphorylase b (EC 2.4.1.1) is converted to a thio-analog of phosphorylase a by phosphorylase kinase, Mg²⁺ and adenosine 5′-O(3-thiotriphosphate)(ATPγS). Conversion proceeds at one-fifth the rate obtained with ATP though the extent of reaction and final level of activation of the enzyme are the same. However, the thiophosphorylase a produced is resistant to phosphorylase phosphatase and, therefore, behaves as a competitive inhibitor with a K_I of 3 μM, similar to the K_M obtained with normal phosphorylase a. ATPγS can also be utilized by protein kinase in the activation of phosphorylase kinase at a rate similar to that obtained with ATP. It is hydrolyzed at 5 to 10 times the normal rate by the sarcoplasmic reticulum ATPase. When added to a muscle glycogen-particulate complex in the presence of Ca²⁺ and Mg²⁺, ATPγS triggers an activation of phosphorylase with simultaneous inhibition of phosphorylase phosphatase as previously observed with ATP.     

Adenosine triphosphate (ATP) hydrolysis promoted by cobalt(III).Participation of polynuclear metal complexes

Complex formation between ATP (adenosine 5′-triphosphate) and tn₂CO^III(aq) (tn = trimethylenediamine) and resulting hydrolysis of the ATP to ADP (adenosine 5′-diphosphate), AMP (adenosine 5′-monophosphate), PP_i (pyrophosphate), and P_i (orthophosphate) have been examined by means of ³¹P nmr. With ATP ～0.1 M and tn₂Co^III(aq) up to 0.3 M, complex formation was promoted by equilibrating solutions for a period at pH 4, after which hydrolysis was allowed to proceed at each of several pHs in the range 5 to 9 prior to quenching by addition of strong base. With ATP 0.01 M and tn₂Co^III(aq) up to 0.08 M, the above procedure was followed in some cases; in other experiments the pH of each ATP/tn₂Co^III(aq) solution was adjusted immediately to a value in the range 5 to 9 with the remainder of the procedure as before. In most cases the hydrolysis was at 25°C, but temperature dependence was also examined. The integrals for the β-phosphorus resonance have been used to analyze for ATP in the quenched solutions; independent measurements of ATP by an enzyme/spectrophotometric method (Bergmeyer) gave similar results. Cobalt to ATP molar ratios up to 1 produce tn₂Co^IIIATP as the predominant ATP complex; this 1:1 complex shows no detectable acceleration in hydrolysis compared to free ATP. Cobalt to ATP molar ratios of ?1 lead to complexes of type (tn₂Co^III)₂ATP and (tn₂Co^III)₃ATP, which exhibit greatly enhanced reactivity towards ATP hydrolysis. At a 2:1 molar ratio (0.1 or 0.01 M ATP), the enhancement is rate is ～10⁵ at pH 7 where the rate is a maximum (comparison for 25°C); at higher molar ratios the rate enhancements are even greater. The results support the view that effective metal ion catalysis of ATP hydrolysis requires formation of reactive species involving more than one metal ion per ATP.     

Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the lld-ACV:bisACV ratio on the flux control

An extended kinetic model for the first two steps of the penicillin biosynthetic pathway in Penicillium chrysogenum is set up. It includes the formation and reduction of the dimer bis--(l--aminoadipyl)-l-cysteinyl-d-valine (bisACV) from the first pathway intermediate lld-ACV and their parallel inhibition of the enzyme ACV synthetase (ACVS). The kinetic model is based on Michaelis-Menten type kinetics, with non-competitive inhibition of the ACVS by both lld-ACV and bisACV, and competitive inhibition of the isopenicillin N synthetase (IPNS) by glutathione. The inhibition constant of lld-ACV, K_ACV is determined to be 0.54 mm. With the kinetic model metabolic control analysis is performed to identify the distribution of rate-control in the pathway at all ratios of lld-ACV:bisACV. It is concluded that the flux control totally resides at the IPNS. This is a result of the regulation of the ACVS by both the lld-ACV and bisACV demanding a higher flux through the IPNS enzyme to alleviate their inhibition. The measurement of an intracellular ratio of lld-ACV:bisACV to be in the range of 1–2 moles per moles emphasises the importance of a fast conversion of lld-ACV to IPN, and accumulation of lld-ACV above the K_m-value of the IPNS should therefore be avoided.     

Reactions of adenosine 5′-phosphorimidazolide with adenosine analogs on a polyuridylic acid template: The uniqueness of the 2′-3′-unsubstituted β-ribosyl system

We have studied the reactions between adenosine 5′-phosphorimidazolide and various adenosine analogs on a poly(U) template. The nucleosides were adenosine (I), 2′-deoxyadenosine (II), 3′-deoxyadenosine (III), 2′-O-methyladenosine (IV), 3′-O-methyladenosine (V), 9-β-d-xylofuranosyladenine (VI), and 9-β-d-arabinofuranosyladenine (VII). We find that the various analogs form triple helices with poly(U) which are of comparable stability, but that only the β-riboside takes part in an efficient template-directed condensation.     

Visualization of drug-nucleic acid interactions at atomic resolution. IV. Structure of an aminoacridine--dinucleoside monophosphate crystalline complex, 9-aminoacridine--5-iodocytidylyl (3'--5') guanosine

9-Aminoacridine forms a crystalline complex with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine (iodoCpG). These crystals are monoclinic, space group P2₁ with $\text{a = 13.98}\text{A}\text{?}\text{, b = 30.58}\text{A}\text{?}\text{, c = 22.47}\text{A}\text{?}$ and β = 113.9 °. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by a combination of Fourier and sum-function Fourier methods. The asymmetric unit contains four 9-aminoacridine molecules, four iodoCpG molecules and 21 water molecules, a total of 245 atoms. 9-Aminoacridine demonstrates two different intercalative binding modes and, along with these, two slightly different intercalative geometries in this model system.The first of these is very nearly symmetric, the 9-amino group lying in the narrow groove of the intercalated base-paired nucleotide structure. The second shows grossly asymmetric binding to the dinucleotide, the 9-amino group lying in the wide groove of the structure. Associated with these two different intercalative binding modes is a difference in geometries in the structures. Although both structures demonstrate C3′ endo (3′–5′) C2′ endo mixed sugar puckering patterns (i.e. both cytidine residues have C3′ endo sugar conformations, while both guanosine residues have C2′ endo sugar conformations), with corresponding twist angles between base-pairs of about 10 °, they differ in the magnitude of the helical screw axis dislocation accompanying intercalation (Sobell et al., 1977a,b). In the pseudosymmetric intercalative structure, this value is about +0.5 Å, whereas in the asymmetric intercalative structure this value is about +2.7 Å. These conformational differences can be best described as a “sliding” of base-pairs on the intercalated acridine molecule.Although the pseudosymmetric intercalative structure can be used in 9-aminoacridine-DNA binding, the asymmetric intercalative structure cannot since this poses stereochemical difficulties in connecting neighboring sugar-phosphate chains to the intercalated dinucleotide. It is possible, however, that the asymmetric binding mode is related to the mechanism of 9-aminoacridine-induced frameshift mutagenesis (Sakore et al., 1977), and we discuss this possibility here in further detail.     

Effects of nucleotide analogs on ATP hydrolysis by P-type ATPases. Comparison between the canine kidney (Na++ K+) ATPase and maize root H+-ATPase

The mechanism by which chemical energy is converted into an electrochemical gradient by P-type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na⁺+ K⁺) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H⁺-ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7-chloro-4-nitrobenz-2-oxa-1.3-diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′-p-fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na⁺+ K⁺) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na⁺+ K⁺) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′-adenylylimidodiphosphate for use as an ATP binding probe.     

Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents

1. o-Iodosobenzoate and 2,2′-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol.2. The inhibition of the Ca²⁺-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2′-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide.3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher K_m for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

A kinetic investigation of the aps-kinase from Chlamydomonas reinhardii CW 15

The reaction kinetics of APS-kinase from Chlamydomonas reinhardii showed that the enzyme formed PAPS from APS upon the addition of ATP. Evidence for a ³⁵S-labelled protein intermediate between APS and PAPS has been obtained. The APS-kinase activity could only be measured in the presence of low concentrations of APS (20 ± 10 μM) and of ATP (0.2 ± 0.05 mM) due to substrate inhibition. The inhibition was partially overcome by low concentrations of 3′,5′-PAP (10,μM). The rates of PAPS formation obtained with cell extracts from the alga varied from 2 to 6 nM PAPS/mg protein/min (33–100 × 10^?12 kat/mg).     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.     

Synthesis and Activity of Modified Cytidine 5′-Monophosphate Probes for T4 RNA Ligase 1

We describe the synthesis of a series of unique base modified ligation probes such as p(5′)C-4-ethylenediamino 3, p(5′)C-4-biotin 4, and pre-adenylated form A(5′)pp(5′)C-4-biotin 6 and tested their biological activity with T4 RNA ligase 1 using a standard pCp probe 1 as a control. The intermolecular ligation assay was developed using a 5′-FAM labeled 24 mer single-stranded (ss) RNA and the average ligation efficiencies for pCp 1, p(5′)C-4-ethylenediamino 3, p(5′)C-4-biotin 4, and pre-adenylated form A(5′)pp(5′)C-4-biotin 6 were found to be 44%, 81%, 39% and 16% respectively, as determined using a denaturing gel analysis. Furthermore, confirmation of the ligation activity of the biotinylated probes to the RNA substrate was confirmed by streptavidin conjugation and analysis by nondenaturing gel electrophoresis. These results strongly suggest that the new probes are valid substrates for T4 RNA ligase 1 and therefore could be useful for developing a miRNA detection system that includes rapid isolation, efficient labeling and detection of miRNAs on sensitivity-enhanced microarrays.     

7‐Deaza‐2′‐Deoxyadenosine‐5′‐Triphosphate as an Alternative Nucleotide for the Pyrosequencing Technology

A new adenosine nucleotide analog suitable for the Pyrosequencing method is presented. The new analog, 7‐deaza‐2′‐deoxyadenosine‐5′‐triphosphate (c⁷dATP), has virtually the same low substrate specificity for luciferase as the currently used analog, 2′‐deoxyadenosine‐5′‐O‐(1‐thiotriphosphate) (dATPαS). The inhibitory effect dATPαS displays on the nucleotide degrading activity of apyrase was reduced significantly by substituting the c⁷dATP for the dATPαS. Both analogs show high stability after long time storage at + 8°C. Furthermore, with the new nucleotide a read length of up to 100 bases was obtained for several templates from fungi, bacteria and viruses.     

Adenosine and gpp(NH)p modulate mouse sperm adenylate cyclase

The possible roles of adenosine and the GTP analogue Gpp(NH)p in regulating mouse sperm adenylate cyclase activity were investigated during incubation in vitro under conditions in which after 30 min the spermatozoa are essentially uncapacitated and poorly fertile, whereas after 120 min they are capacitated and highly fertile. Adenylate cyclase activity, assayed in the presence of 1 mM ATP and 2 mM Mn²⁺, was determined by monitoring cAMP production. When adenosine deaminase (1 U/ml) was included in the assay to deplete endogenous adenosine, enzyme activity was decreased in the 30-min suspensions but increased in the 120-min samples (P < 0.02). This suggests that endogenous adenosine has a stimulatory effect on adenylate cyclase in uncapacitated spermatozoa but is inhibitory in capacitated cells. Since the expression of adenosine effects at low nucleoside concentrations usually requires guanine nucleotides, the effect of adding adenosine in the presence of 5 x 10^–5 M Gpp(NH)p was examined. While either endogenous adenosine or adenosine deaminase may have masked low concentration (10^?9?10^?7 M) effects of exogenous adenosine, a marked inhibition (P < 0.001) of adenylate cyclase activity in both uncapacitated and capacitated suspensions was observed with higher concentrations (>10^?5 M) of adenosine. Similar inhibition was also observed in the absence of Gpp(NH)p, suggesting the presence of an inhibitory P site on the enzyme. In further experiments, the effects of Gpp(NH)p in the presence and absence of adenosine deaminase were examined. Activity in 30-min suspensions was stimulated by the guanine nucleotide and in the presence of adenosine deaminase this stimulation was marked, reversing the inhibition seen with adenosine deaminase alone. In capacitated suspensions the opposite profile was observed, with Gpp(NH)p plus adenosine deaminase being inhibitory; again, this was a reversal of the effects obtained in the presence of adenosine deaminase alone, which had stimulated enzyme activity. These results suggest the existence of a stimulatory adenosine receptor site (R_a) on mouse sperm adenylate cyclase that is expressed in uncapacitated spermatozoa and an inhibitory receptor site (R_i) that is expressed in capacitated cells, with guanine nucleotides modifying the final response to adenosine. It is concluded that adenosine and guanine nucleotides may regulate mouse sperm adenylate cyclase activity during capacitation.     

The reaction of 14C-labelled platinum ethylenediamine dichloride with adenine compounds and DNA

Various derivatives of adenine have been studied with regard to their rate of reaction with ¹⁴C-labelled platinum ethylenediamine dichloride, Pt(¹⁴C-en)Cl₂. The reactivities have been calculated from the “rate of disappearance” of Pt(¹⁴C-en)Cl₂ using chromatographic separation of reactants and products.Adenine and adenosine react very slowly at 37° whereas other adenine derivatives react much more readily in the order: poly A > AMP > ApA > poly d(AT). From the numerical values of the rate constants it is concluded that the presence of a phosphate group increases the reaction rate considerably. This is partly the explanation of the rapid reaction of poly A which possesses terminal phosphate groups. However adjacent adenine moieties such as those in polyadenylic acid (poly A) and adenosyl-3′5′-adenosine (ApA) may also react by another mechanism which involves the 6-NH₂ groups.The energies of activation of the second order reaction with platinum ethylenediamine dichloride (PtenCl₂) are 12.9, 18.8, 19.0 kcal/mole for poly A, AMP and ApA respectively.In DNA, no free phosphate groups are present, and the occurrence of adjacent adenines will be low. The reaction of PtenCl₂ with DNA seems to involve a rapid attack on deoxyguanosine (GdR) and a slow reaction with deoxyadenosine (AdR) and deoxycytidine (CdR).     

Studies on the synthesis of CDP-diacylglycerol: Stimulation by GTP and inhibition by ATP and fluoride

The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Occurrence of a 3′-phosphoadenosine 5′-Phosphosulphate synthesizing system In two Ochromonas species

The presence of an enzyme capable of incorporating ³⁵SO₄^2? into 3′-phosphoadenosine 5′-phosphosulphate has been demonstrated,in Ochromonas danica and O. malhamensis. This system probably includes the enzymes ATP:sulphate adenyltransferase. E.C. 2.7.7.4 and ATP:adenylsulphate 3′-phosphotransferase, E.C. 2.7.1.25.