Conversion of adenosine(5') oligophospho(5') adenosines into inosine(5') oligophospho(5') inosines by non-specific adenylate deaminase from the snail Helix pomatia.

Until now, the catabolism of adenosine(5')triphospho(5')adenosine (Ap3A) and adenosine(5')tetraphospho(5')adenosine (Ap4A) has been thought to commence with either hydrolytic or phosphorolytic cleavage of their oligophosphate chains, depending on the organism. Here, we show that in the extracts from the retractile 'foot' of the snail Helix pomatia deamination predominates; the adenosine moieties of these and other adenosine(5')oligophospho(5')adenosines (ApnAs) undergo successive deamination leading, via an inosine(5')oligophospho(5')adenosine (IpnA), to the corresponding inosine(5')oligophospho(5')inosine (IpnI). The reactions are catalyzed by the non-specific adenylate deaminase described earlier (Stankiewicz, A.J. (1983) Biochem. J. 215, 39-44). We describe TLC and HPLC systems which allow the separation of any of the deaminated derivatives from its parent compound; Ap2A, Ap3A, Ap4A or Ap5A. The Km values for these substrates are 20, 22, 32 and 39 microM, respectively, whereas the Km for 5'-AMP is 12 microM. Relative substrate specificities for these compounds amount to 25, 18, 14, 7 and 100. The enzyme was shown also to deaminate phosphonate and thiophosphate analogues of Ap3A.

2',3'-dideoxynucleoside triphosphates (ddNTP) and di-2',3'-dideoxynucleoside tetraphosphates (ddNp4ddN) behave differently to the corresponding NTP and Np4N counterparts as substrates of firefly luciferase, dinucleoside tetraphosphatase and phosphodiesterases.

2',3'-Dideoxynucleosides (ddN) and their derivatives are currently used as antiretroviral compounds. Their active agents are the corresponding 2',3'-dideoxynucleoside triphosphates (ddNTPs) generated inside the cell by host kinases. Dinucleoside tetraphosphates (Np4Ns) are molecules of interest in metabolic regulation; their synthesis in vitro can be catalyzed by firefly luciferase. The relative synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate or adenosine(5')tetraphospho(5')adenosine (Ap4A) from ATP is about 100-fold faster than that of di-2',3'-dideoxyadenosine 5',5'''-P1,P4-tetraphosphate or 2',3'-dideoxyadenosine (5')tetraphospho (5')-2',3'-dideoxyadenosine (ddAp4ddA) from ddATP. In the presence of ATPgammaS and ddATP the yield of adenosine(5')tetraphospo(5')-2',3'-dideoxyadenosine (Ap4ddA) was similar to that attained for Ap4A in the presence of ATP. The findings of this work indicate that the presence of a 3'-hydroxyl group is essential for the formation of the luciferase-luciferin-AMP complex, and explains the very low yield of ddAp4ddA in the presence of luciferase, luciferin and ddATP. The absence of 3'-hydroxyl groups in ddAp4ddA greatly hindered their hydrolysis by snake venom phosphodiesterase, asymmetrical dinucleoside tetraphosphatase and by a purified membrane preparation from rat liver. The possibility of using di-2',3'-dideoxynucleoside tetraphosphate (ddNp4ddN) or nucleoside(5')tetraphospho(5')-2',3'-dideoxynucleoside (Np4ddN) as a source of the active retroviral agent ddNTP, for example in HIV infection, is outlined.

Purification and properties of three proteases from the larvae of the brine shrimp Artemia salina.

Three proteases, termed A, B and C, have been characterized and partially purified from Artemia salina larvae. Enzyme A is active on benzyloxycarbonyl L-leucin p-nitrophenyl ester (Km = 53 micron, determined at pH 8 and 37 degrees C) but not on alpha-N-benzoyl-DL-arginine p-nitroanilide and is strongly inhibited by micron concentrations of phenylmethylsulfonylfluoride. Enzymes B and C are active on alpha-N-benzoyl-L-arginine p-nitroanilide (Km = 20 and 11 micron, respectively) but not on benzyloxycarbonyl-L-leucine p-nitrophenyl ester and B, but not C, is inhibited by mM concentrations of phenylmethylsulfonyfluoride. Enzymes A, B and C are optimally active at alkaline pH values, do not require either metal ions or -SH groups for their catalytic activity and have molecular weights of 38 000, 33 000 and 34 000, respectively. After heating for 5 min at pH 7.5 and in the presence of 0.7 M KCl half inactivation of proteases A, B and C was attained at 60, 52 and 45 degrees C, respectively.

Occurrence of adenosine 2',5'-bisphosphate in rat liver.

Adenosine 2',5'-bisphosphate (pAp) is present in liver from 2-day-fasted rats, at a concentration of around 1 microM. pAp was obtained through perchloric acid extraction of the liver followed by two successive DEAE-cellulose chromatographies and an ion-pair high-pressure liquid chromatography. Both pAp extracted from liver and that obtained from a commercial source showed the same pattern of hydrolysis by alkaline phosphatase, i.e., more 5'-AMP than 2'-AMP was obtained as an intermediate of the reaction.

Particulate diadenosine 5',5"'-P1,P3-triphosphate hydrolases in rat brain: two specific dinucleoside triphosphatases and two phosphodiesterase I-like hydrolases.

Rat liver and brain differ in the distribution pattern of the total hydrolytic activity on diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) between the soluble and particulate fractions. The Ap3A-hydrolase activity in both the soluble and particulate liver fractions and in the brain soluble fraction had been previously studied in detail. We report now on the brain particulate fraction which, unlike liver, showed a low unspecific phosphodiesterase I-like (PDEaseI, EC 3.1.4.1) activity relative to the specific dinucleoside triphosphatase (Ap3Aase, EC 3.6.1.29). Two PDEaseI-like forms (PDEaseI-A and PDEaseI-B), with different apparent Mrs and kinetic properties, and two Ap3Aases (Ap3Aase-alpha and Ap3Aase-beta) were solubilized with 0.5% Triton X-100 from the particulate fraction. Ap3Aase-alpha resembled the cytosolic Ap3Aase (Ap3Aase-c), a known situation in liver. Comparative to Ap3Aase-alpha, Ap3Aase-beta showed a slightly higher Km (35 vs. 15 micron) and lower isoelectric point (5.25 vs. 5.45); Ap3Aase-beta was absent from the soluble fraction, and its recovery was unaffected by proteinase inhibitors, strongly arguing for distinct soluble and particulate turnover pathways for dinucleoside polyphosphates.

Dinucleosidasetetraphosphatase in rat liver and Artemia salina.

A comparative study of an enzymatic activity present in Artemia salina and rat liver which specifically splits dinucleoside tetraphosphates is presented. All the purine and pyrimidine dinucleoside tetraphosphates tested, i.e. diadenosine, diguanosine, dixanthosine and diuridine tetraphosphates, were substrates of both enzymes with similar maximum velocities and Km values, (around 10 muM). The inhibition by nucleotides of the enzyme from the two sources is also similar. Particularly relevant is the strong inhibition caused by nucleoside tetraphosphates which have Ki values in the nanomolar range. The Artemia enzyme has a slightly lower molecular weight (17 500) than the liver enzyme (21 000) and is more resistant to acidic pH. Based on previous findings, the enzyme from Artemia salina was named diguanosinetetraphosphatase (EC 3.6.1.17) by the Enzyme Commission. The results presented in this paper show that the liver and Artemia enzymes are similar, and we propose to name this enzyme as dinucleosidetetraphosphatase or dinucleoside-tetraphosphate nucleotidehydrolase.

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases.

The findings presented here originally arose from the suggestion that the synthesis of dinucleoside polyphosphates (Np(n)N) may be a general process involving enzyme ligases catalyzing the transfer of a nucleotidyl moiety via nucleotidyl-containing intermediates, with release of pyrophosphate. Within this context, the characteristics of the following enzymes are presented. Firefly luciferase (EC 1.12. 13.7), an oxidoreductase with characteristics of a ligase, synthesizes a variety of (di)nucleoside polyphosphates with four or more inner phosphates. The discrepancy between the kinetics of light production and that of Np(n)N synthesis led to the finding that E*L-AMP (L = dehydroluciferin), formed from the E*LH(2)-AMP complex (LH(2) = luciferin) shortly after the onset of the reaction, was the main intermediate in the synthesis of (di)nucleoside polyphosphates. Acetyl-CoA synthetase (EC 6.2.1.1) and acyl-CoA synthetase (EC 6.2.1. 8) are ligases that synthesize p(4)A from ATP and P(3) and, to a lesser extent, Np(n)N. T4 DNA ligase (EC 6.5.1.1) and T4 RNA ligase (EC 6.5.1.3) catalyze the synthesis of Np(n)N through the formation of an E-AMP complex with liberation of pyrophosphate. DNA is an inhibitor of the synthesis of Np(n)N and conversely, P(3) or nucleoside triphosphates inhibit the ligation of a single-strand break in duplex DNA catalyzed by T4 DNA ligase, which could have therapeutic implications. The synthesis of Np(n)N catalyzed by T4 RNA ligase is inhibited by nucleoside 3'(2'),5'-bisphosphates. Reverse transcriptase (EC 2.7.7.49), although not a ligase, catalyzes, as reported by others, the synthesis of Np(n)ddN in the process of removing a chain termination residue at the 3'-OH end of a growing DNA chain.

Time course of luciferyl adenylate synthesis in the firefly luciferase reaction.

The time course of luciferyl adenylate formation in the reaction catalyzed by firefly luciferase (EC 1.13.12.7) has been followed. The properties of luciferyl adenylate, enzymatically or chemically synthesized, as substrate of luciferase, have been compared. The potential use of luciferyl adenylate for luciferase detection is here proposed.

Firefly luciferase synthesizes P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and other dinucleoside polyphosphates.

The synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) has been considered, for a long time, to be catalyzed mainly by some aminoacyl-tRNA synthetases [Brevet et al. (1989) Proc. Natl. Acad. Sci. USA 86, 8275-8279]. Recently, yeast Ap4A phosphorylase, acting in reverse (Guranowski et al. (1988) Biochemistry 27, 2959-2964), was shown to synthesize Ap4A, too. In the case of the synthetases, the intermediate complex E-aminoacyl-AMP may serve as donor of AMP to ATP, yielding Ap4A. Here we demonstrate that firefly luciferase (EC 1.13.12.7) which forms the E-luciferin-AMP intermediate also synthesizes Ap4A as well as other dinucleoside polyphosphates. We suggest moreover that: other enzymes (mainly synthetases and some transferases), which catalyze the transfer of a nucleotidyl moiety, via nucleotidyl-containing intermediates and releasing PPi may produce dinucleoside polyphosphates.

Location of dinucleoside triphosphatase in the matrix space of rat liver mitochondria.

The submitochondrial location of dinucleoside triphosphatase (EC 3.6.1.29), previously shown to be in part associated with mitochondria, has been studied in rat liver. The precipitability and latency of activity in organelle suspensions, and the profile of solubilization by digitonin, were like those of the matrix space marker glutamate dehydrogenase, and differed from those of other submitochondrial fractions. This, and the synthesis of diadenosine polyphosphates by mitochondrial aminoacyl-tRNA synthetases, suggest the occurrence of a pathway for the intramitochondrial turnover of diadenosine 5',5'''-P1,P3-triphosphate (Ap3A).

Dehydroluciferyl-AMP is the main intermediate in the luciferin dependent synthesis of Ap4A catalyzed by firefly luciferase.

It was previously assumed that E x LH2-AMP was the intermediate complex in the synthesis of Ap4A catalyzed by firefly luciferase (EC 1.13.12.7), when luciferin (LH2) was used as cofactor. However, here we show that LH2 is partly transformed, shortly after the onset of the luciferase reaction, to dehydroluciferin (L) with formation of an E x L-AMP complex which is the main intermediate for the synthesis of Ap4A. Formation of three more derivatives of LH2 were also observed, related to the production of light by the enzyme. CoA, a known stimulator of light production, inhibits the synthesis of Ap4A by reacting with the E x L-AMP complex and yielding L-CoA.

T4 DNA ligase synthesizes dinucleoside polyphosphates.

T4 DNA ligase (EC 6.5.1.1), one of the most widely used enzymes in genetic engineering, transfers AMP from the E-AMP complex to tripolyphosphate, ADP, ATP, GTP or dATP producing p4A, Ap3A, Ap4A, Ap4G and Ap4dA, respectively. Nicked DNA competes very effectively with GTP for the synthesis of Ap4G and, conversely, tripolyphosphate (or GTP) inhibits the ligation of DNA by the ligase. As T4 DNA ligase has similar requirements for ATP as the mammalian DNA ligase(s), the latter enzyme(s) could also synthesize dinucleoside polyphosphates. The present report may be related to the recent finding that human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5''-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29).

Acyl-CoA synthetase catalyzes the synthesis of diadenosine hexaphosphate (Ap6A).

The synthesis of diadenosine hexaphosphate (Ap6A), a potent vasoconstrictor, is catalyzed by acyl-CoA synthetase from Pseudomonas fragi. In a first step AMP is transferred from ATP to tetrapolyphosphate (P4) originating adenosine pentaphosphate (p5A) which, subsequently, is the acceptor of another AMP moiety from ATP generating diadenosine hexaphosphate (Ap6A). Diadenosine pentaphosphate (Ap5A) and diadenosine tetraphosphate (Ap4A) were also synthesized in the course of the reaction. In view of the variety of biological effects described for these compounds the potential capacity of synthesis of diadenosine polyphosphates by the mammalian acyl-CoA synthetases may be relevant.

A low Km nucleoside 3'(2'),5-bisphosphate 3'(2')-phosphohydrolase from rat liver.

In the course of an investigation on the occurrence in rat liver of a specific hydrolytic activity on adenosine 2',5'-bisphosphate, a nucleoside 3'(2'),5'-bisphosphate 3'(2')-phosphohydrolase was purified following standard procedures. The enzyme hydrolyzes the phosphate group joined to the 3' or the 2' position of the following nucleotides (relative velocities indicated in brackets): PAdoP (100), PCydP (95), PGuoP (80), PAdo2'P (40), PdAdoP (4), SPAdoP (18). Other nucleotides were not substrates of the reaction: NADP+, PAdoPP, PPGuoP, AdoP, PAdo, GuoP, PGuo, ADP, ATP, cAMP, adenosine(3')phospho(5')adenosine. The Km values determined for PAdoP and PAdo2'P were 10 and 7 microM, respectively. Two isoforms were separated by chromatography on a Mono Q column. Both isoforms were kinetically indistinguishable, presenting a pI value of 5.35, a molecular mass of 38 kDa, pH optimum of 8.0, and strictly required Mg2+ or Mn2+. An enzymatic activity similar to the one described here has already been reported in guinea pig liver [5]. These authors however only obtained 1 enzymatic form with Km values of 3.1 and 1.8 mM for PAdoP and PAdo2'P, respectively. The potential physiological role of this enzyme in the metabolism of sulphate is also considered. The previously registered number EC 3.1.3.7 could be applied to this activity.

Diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A), ATP and catecholamine content in bovine adrenal medulla, chromaffin granules and chromaffin cells.

The level of diadenosine 5',5"-P1-P4-tetraphosphate (diadenosine tetraphosphate or Ap4A), catecholamines, ATP and other nucleotides has been investigated in perchloric acid extracts of bovine adrenal medulla, chromaffin granules and cultured chromaffin cells. As a control, the amount of Ap4A and ATP has also been measured in human blood platelets. The following values (nmol/mg protein) were found in adrenal medulla: Ap4A, 0.019 +/- 0.004; ATP, 109 +/- 11; ADP, 23.8 +/- 5.8; AMP, 11.3 +/- 1.5; p4A, 0.18 +/- 0.08; catecholamines, 460 +/- 57. The level of Ap4A, catecholamines and ATP (nmol/mg protein) found in chromaffin granules and in chromaffin cells were, respectively: (0.15 +/- 0.07; 2175 +/- 99; 531 +/- 66) and (0.22 +/- 0.14; 1143 +/- 277; 222 +/- 53). In all the cases investigated, the ratio catecholamines/ATP and catecholamines/Ap4A were around 5 and in the order of 10(3), respectively. The amount of Ap4A found here, in bovine adrenal medulla, chromaffin granules and chromaffin cells, is two orders of magnitude lower than previously reported.

Inhibition and activation of enzymes. The effect of a modifier on the reaction rate and on kinetic parameters.

A combined analysis of enzyme inhibition and activation is presented, based on a rapid equilibrium model assumption in which one molecule of enzyme binds one molecule of substrate (S) and/or one molecule of a modifier X. The modifier acts as activator (essential or non-essential), as inhibitor (total or partial), or has no effect on the reaction rate (v), depending on the values of the equilibrium constants, the rate constants of the limiting velocity steps, and the concentration of substrate ([S]). Different possibilities have been analyzed from an equation written to emphasize that v = f([X]) is, in general and at a fixed [S], a hyperbolic function. Formulas for Su (the value of [S], different from zero, at which v is unaffected by the modifier) and v(su) (v at that particular [S]) were deduced. In Lineweaver-Burk plots, the straight lines related to different [X] generally cross in a point (P) with coordinates (Su, v(su)). In certain cases, point P is located in the first quadrant which implies that X acts as activator, as inhibitor, or has no effect, depending on [S]. Furthermore, we discuss: (1) the apparent Vmax and Km displayed by the enzyme in different situations; (2) the degree of effect (inhibition or activation) observed at different concentrations of substrate and modifier; (3) the concept of Ke, a parameter that depends on the concentration of substrate and helps to evaluate the effect of the modifier: it equals the value of [X] at which the increase or decrease in the reaction rate is half of that achieved at saturating [X]. Equations were deduced for the general case and for particular situations, and used to obtain computer-drawn graphs that are presented and discussed. Formulas for apparent Vmax, Km and Ke have been written in a way making it evident that these parameters can be expressed as pondered means.

An easy procedure to transform the ratio of two polynomials of first degree into Michaelis-Menten-type equations. Application to the ordered Uni Bi enzyme mechanism.

It is not always clear that some equations affected by complicated factors can, actually, be interpreted as a ratio of two polynomials of first degree and so that they can be, in general, represented by rectangular hyperbolas. In this paper we present an easy procedure to rearrange those equations into Michaelis-Menten-type equations and so to make the aspects of these rectangular hyperbolas more clear, particularly for researchers familiar with general biochemistry. As an example, the method is applied to transform the classical rate equation of the Cleland's Ordered Uni Bi enzyme mechanism.

Theoretical isoelectric points and electric charges of mutated human hemoglobin subunits.

Isoelectric point values have been determined from a theoretical standpoint for alpha, beta, gamma and delta human abnormal subunits bearing elimination or addition of 1-2 different aminoacids with acid-base residues. The charge distribution, in relation to pH values, of the four types of subunits are also reported. These values may be of interest both to predict the electrophoretic behaviour and to characterize human abnormal hemoglobins.

Labeled adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')tetraphospho(5')nucleoside (Ap4N). Synthesis with firefly luciferase.

Labeled dinucleoside polyphosphates are not commercially available, in spite of being important molecules in metabolic regulation. Firefly luciferase (EC 1.13.12.7) is a useful enzyme for the synthesis of adenosine(5')tetraphospho(5')adenosine (Ap4A). As luciferase behaves as a nucleotidase at low ATP concentration, adequate concentrations (higher than 0.1 mM ATP) should be used to obtain a good yield of labeled Ap4A. [32P]Ap4A has also been synthesized from ATP and [32P]PPi. In a first step, [beta, gamma-32P]ATP is generated in a ATP-[32P]PPi exchange reaction catalyzed by luciferase. In a second step, the reaction is supplemented with pyrophosphatase and 32P labeled Ap4A is obtained. Radioactive adenosine(5')tetraphospho(5')nucleoside (Ap4N) can also be synthesized from ATP gamma S and labeled NTP or from low concentrations of labeled ATP and high concentrations of cold NTP. The syntheses of radioactive ApnA and pnA (n > 4) can also be approached with luciferase.

A program to calculate the isoelectric point of macromolecules.

A program containing 260 sentences, written in BASIC and adapted to be run in personal computers, has been developed to calculate isoelectric point values of macromolecules (proteins, nucleic acids, polysaccharides, etc.). This implies the calculation of the coefficients and roots of a particular kind of polynomial. With common personal computers and using a compiled version of the program, the time required to calculate pI values of macromolecules containing for example, 9, 12, and 18 acid-base residues with different pK values was around 10 s, 2 min, and 2 h respectively.