Adenylate Deaminase Research Articles

Adenylate Deaminase (5′‐Adenylic Acid Deaminase, AMPDA)‐Catalyzed Deamination of 5′‐Deoxy‐5′‐Substituted and 5′‐Protected Adenosines: A Comparison with the Catalytic Activity of Adenosine Deaminase (ADA)

AbstractThe enzyme adenylate deaminase (AMPDA) is able to catalyze the hydrolytic deamination of 5′‐substituted and 5′‐protected 5′‐deoxyadenosines, whereas limited or no activity is shown by adenosine deaminase (ADA) towards the same substrates. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Deamination of 5′-substituted-2′,3′-isopropylidene adenosine derivatives catalyzed by adenosine deaminase (ADA, EC 3.5.4.4) and complementary enzymatic biotransformations catalyzed by adenylate deaminase (AMPDA, EC 3.5.4.6): a viable route for the preparation of 5′-substituted inosine derivatives

Adenosine deaminase (ADA) catalyzes the deamination of 2′,3′-isopropylidene adenosine and the corresponding 5′-amino derivative in a 3% dimethylsulfoxide aqueous solution. Whereas ADA is unable to convert other 5′-substituted derivatives (acetate, acetamido, azide), the enzyme adenylate deaminase (AMPDA) accepts all the above compounds as substrates for their biotransformation to the corresponding 5′-substituted inosine derivatives.

Blood purine and energy status in rats with colitis.

Colitis reduces the blood and tissue levels of adenosine deaminase and adenylate deaminase. Whether this has any effect on blood purines remains to be determined. The aim of this study was to measure the adenylate pool, substrates of the above enzymes, and energy status in blood from rats with colitis. Colitis was induced by intrarectal administration of acetic acid and followed over a period of seven days. The levels of ATP, ADP, AMP, adenosine, inosine, and uric acid were analyzed by HPLC, and energy status was estimated. Myeloperoxidase was used as a marker of colitis. Concentrations of ATP, ADP, AMP and adenosine decreased during days 1-5, whereas energy status decreased on day 2. The concentrations of inosine, uric acid, and hemoglobin remained unaltered, whereas colonic myeloperoxidase activity increased. These, findings demonstrate colitis-induced reduction of the circulating purines, which may be due to their enhanced usage for the repair of the inflamed colon.

Vitamin A Deficiency in Mice Enhances the Colonic Level of Purine Enzyme Activity

Background: Vitamin A is an important nutritional factor that regulates normal growth and functions of epithelial cells of the gastrointestinal tract. Objective: The objective of this study was to examine the role of vitamin A on the histological and biochemical changes in the colon of mice. Methods: To address this issue, vitamin A deficiency (VAD) was developed in mice by placing them on a VAD diet from weaning up to 120–170 days. Infiltration of inflammatory cells in the colon was determined histologically. Activities of adenosine deaminase, adenylate deaminase, purine nucleoside phosphorylase and myeloperoxidase were determined. Results: VAD in mice induced a significant increase in the number of mast cells per 100 crypts. There was also an abundance of other connective tissue cells such as plasma cells, lymphocytes and neutrophils around the crypts in the lamina propria. The colonic activity of adenosine deaminase and adenylate deaminase was increased due to VAD, whereas purine nucleoside phosphorylase activity remained unchanged. Immunohistochemical analysis showed an increased expression of adenosine deaminase in VAD mice colon. The increase in myeloperoxidase activity was not statistically significant. Conclusions: VAD causes upregulation of purine enzyme, which together with an increased number of inflammatory cells might exacerbate colonic injuries in VAD condition.

Toxicity and mutagenicity studies of DN-50000 ® and RP-1 ® enzymes

Improved yields of 5′-nucleotides from yeast extract for food flavouring purposes is possible through use of microbial nucleotidases, which will be available to food processors as the flavour enhancer Aromild ®. The safety of these enzymes, 5′-phosphodiesterase (RP-1 ®) and the 5′-adenylic deaminase (DN-50000 ®) was investigated in male and female rats. Feeding rats a diet admixed with 500, 2000 and 8000 mg/kg body weight of DN-50000 for 35 days resulted in no significant dose-related changes in body weight, water consumption, urinalysis, haematological profiles, blood chemistry or histopathological profiles of either male or female rats from consumption of the enzyme preparation. There was an increase in the absolute and/or relative organ weights of the submaxillary (salivary) glands in both sexes at 8000 mg/kg. The no-observed-effect level (NOEL) for DN-50000 was clearly evident at 2000 mg/kg. Consumption of RP-1 enzyme for 35 days at dietary levels of 500, 2000 and 8000 mg/kg body weight resulted in no significant changes in the above mentioned parameters, which could be directly attributed to a dose-related effect, with the exception of an increase in the absolute and relative weights of submaxillary glands of both male and female rats in the 2000 and 8000 mg/kg groups. The increase in weight of the submaxillary glands was associated with histological evidence of acinar cell hypertrophy. The NOEL for dietary consumption of RP-1 was clearly evident at 500 mg/kg. In a follow-up study in which rats were gavaged with 2000 mg/kg RP-1, submaxillary gland hypertrophy did not occur. These studies suggest that DN-50000 and RP-1 exert an action on submaxillary glands similar to that which has been previously reported for the enzyme pancreatin. Neither DN-50000 nor RP-1 were mutagenic in the Ames assay using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 or Escherichia coli strain WP2uvrA, in the presence or absence of S9 mix.

Treatment of Experimental Autoimmune Encephalomyelitis With Adenylate Deaminase From Penicillium lanoso-viride

The effect of intramuscularly administered immunomodulator, adenylate deaminase (E.C. 3.5.4.6), fromPenicillium lanoso-viride on the clinical score of acute experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease, was examined by inoculation of guinea pigs with rabbit brain and spinal cord homogenate (encephalitogen) and complete Freund's adjuvant. Adenylate deaminase (ADA) was effective in delaying the onset of clinical disease. ADA inhibited the severity of EAE. There was a significant decrease in clinical signs. A decrease in the number of morbid and dead animals was observed. Of ADA treated animals, 50–80% developed no clinical manifestations of EAE. The optimal version of treatment was a single preventive injection of ADA 1 day before the sensitization and then every second day after immunization for 20 days. ADA treatment of immunized animals diminished the activity of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase in the cerebrospinal fluid, as well the amount of complement fixing antiencephalitogenic antibodies in the blood serum. The mechanism of ADA cerebroprotective action is discussed. Significant skin-allergic cross-reaction of delayed-type hypersensitivity between ADA and encephalitogen was observed.

The occurrence and properties of 5'-AMP deaminase in dried and toasted nori

SUMMARY: Crude 5′-adenylic acid deaminase (5′-AMP deaminase), extracted from hoshi-nori (dried nori) and yaki-nori (toasted nori), was investigated. Substantial activity of 5′-AMP deaminase was found in the extracts of both dried and toasted nori, but the activity of the latter was lower than the former. The properties of the enzyme were as follows. The optimum pH and optimum temperature were observed in the ranges of pH 7.0–8.0 and 30–50°C, respectively. Metal ions Ca2+ and Sr2+ were most effective on the activation of enzyme, while Cu2+ and Fe2+ did not influence the activity. Ca2+ was also effective for thermal stability of the enzyme. The enzyme activity was comparatively stable even at 50°C in the presence of 0.1 M Ca2+, while it was completely inactivated at 50°C in the absence of Ca2+. The enzyme was specific only to 5′-AMP. The other adenylic derivatives such as 3′-AMP, 5′-ADP, and 5′-ATP were not reacted. The Km value for 5′-AMP was estimated to be 2.77 mM. Addition of p-CMB, an inhibitor of SH-enzyme, to the reaction mixture inhibited the enzyme activity. Half inhibition was observed with 5 mM p-CMB concentration. The properties of the enzyme were not essentially different between dried and toasted nori. The activity of the latter was lower than that of the former, and excess toasting (180°C 15 s) caused a remarkable loss of activity.

Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors.

1. The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP-activated inward currents at rat P2X(1-4) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2. Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5'-adenylic deaminase, and purified using reverse-phase chromatography. 3. Against ATP-responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)>Ip4I (6.3)>Ip3I (>4.5). Ip5I (10-100 nM) caused a concentration-dependent rightwards displacement of the ATP concentration-response curve without reducing the maximum ATP effect. However, the Schild plot was non-linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non-surmountable antagonist. 4. Against ATP-responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6. 0)>Ip5I (5.6)>Ip3I (>4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations. 5. Diinosine polyphosphates failed to inhibit ATP-responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1 - 4 receptors. 6. Thus, Ip5I acted as a potent and relatively-selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high-affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (</=100 nM) concentrations but has more complex actions at higher (>100 nM) concentrations.

9-(2-Phosphonylmethoxyethyl)- N6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) as a prodrug of 9-(2-Phosphonylmethoxyethyl)guanine (PMEG)

9-(2-Phosphonylmethoxyethyl)- N 6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) is an acyclic nucleotide analog of the [9-(2-phosphonylmethoxyethyl)-] (PME) series containing a cyclopropyl substituent on the N 6 position of the 2,6-diaminopurine (DAP) base. Growth inhibition assays in a broad range of tumor cell lines demonstrated that this analog had potent antiproliferative activity with ic 50 values similar to those of the structurally related guanine analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG). A substantially lower growth inhibitory effect was observed for the 2,6-diaminopurine analog, PMEDAP. To dissect the basis for these varying potencies, the metabolism of the three analogs was examined in a human pancreatic carcinoma cell line, BxPC-3. HPLC analysis of the intracellular metabolites demonstrated that the cpr-PMEDAP was deaminated to PMEG and subsequently phosphorylated to PMEG mono- and diphosphates (PMEGp and PMEGpp). The level of PMEGpp generated from cpr-PMEDAP-treated cells was 50% greater than the level generated from cells incubated with PMEG. The presence of PMEG in the DNA of cells incubated with cpr-PMEDAP confirmed that the cpr-PMEDAP was converted to PMEG. In contrast, PMEDAP was not deaminated to PMEG, but directly phosphorylated to PMEDAPp and PMEDAPpp. The adenylate deaminase inhibitor 2′-deoxycoformycin (dCF) inhibited the conversion of cpr-PMEDAP in a rat liver cytosolic extract and increased the ic50 value for growth inhibition by 40-fold. The antiproliferative activities of PMEG and PMEDAP were unaffected by dCF. Thus, it appears that cpr-PMEDAP, but not PMEDAP, is converted by an adenylate deaminase-like enzyme and functions as a prodrug of PMEG.

N 6-cyclopropyl-PMEDAP: a novel derivative of 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) with distinct metabolic, antiproliferative, and differentiation-inducing properties

N 6-Cyclopropyl-PMEDAP (cPr-PMEDAP) is a novel derivative of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP). Its cytostatic activity was found to be 8- to 20-fold more pronounced than that of PMEDAP and equivalent to that of the guanine derivative 9-(2-phosphonylmethoxyethyl)guanine (PMEG) against a variety of tumor cell lines. Unlike PMEDAP, but like PMEG, cPr-PMEDAP was equally cytostatic to wild-type and 9-(2-phosphonylmethoxyethyl)adenine/PMEDAP-resistant variants of the human erythroleukemia K562 and the murine leukemia L1210 cell lines. Also, cPr-PMEDAP and PMEG proved to be equipotent inducers of K562 and rat choriocarcinoma RCHO cell differentiation, whereas the differentiation-inducing activity of PMEDAP was 5- to 25-fold less pronounced. Furthermore, compared to PMEDAP, cPr-PMEDAP and PMEG were 10- to 25-fold more potent in inhibiting the progression of K562 cells through the S phase of the cell cycle, resulting in a marked accumulation of the four 2′-deoxyribonucleoside 5′-triphosphate pools. The biological effects of cPr-PMEDAP, but not PMEDAP, were reversed by the adenylate deaminase inhibitor 2′-deoxycoformycin (dCF). Formation of the deaminated derivative of cPr-PMEDAP (i.e. PMEG) was demonstrated in crude extracts from K562 and L1210 cells and in metabolism studies with radiolabeled cPr-PMEDAP and PMEG. This is the very first example of an acyclic nucleoside phosphonate analogue that is susceptible to deamination. However, cPr-PMEDAP was not recognized as a substrate by purified adenosine deaminase or by adenylate deaminase. These findings might point to an as yet unidentified cellular enzyme, sensitive to dCF but different from the common adenosine and AMP deaminases. Our data demonstrate the superior antiproliferative and differentiation-inducing effects of cPr-PMEDAP on tumor cells, as compared to the parent compound PMEDAP, based on the unique metabolic properties of this novel compound.

Studies on purine enzymes in experimental colitis.

Although the role of adenosine deaminase (ADA), adenylate deaminase (AMP-DA), purine nucleoside phosphorylase (PNP) is well documented in gastric and intestinal carcinoma, their role in inflammatory bowel diseases remains unknown. In the present study, we investigated the profile of these enzymes in blood and intestinal tissues during colitis. Colitis induced in Wistar rats by acetic acid was monitored by a marker enzyme myeloperoxidase (MPO). The tissue levels of MPO increased on 1, 2, 5 and 6 days post-administration (PA) of acetic acid and declined to the control levels by day 7 PA. In parallel the blood levels of ADA and AMP-DA decreased on days 1, 2 and 5 without any significant change on days 6 and 7 PA. Similar observations were recorded for these enzymes in the cytosolic extracts of colonic tissue specimens. In contrast, PNP remained unaltered in both blood and tissue samples. These findings suggest an inverse-relationship between inflammation and purine deaminases in both blood and tissues.

Purification and Characterization of NAD:Penicillamine ADP Transferase from Bacillus sphaericus: A NOVEL NAD-DEPENDENT ENZYME CATALYZING PHOSPHORAMIDE BOND FORMATION

A strain of Bacillus sphaericus isolated from a local soil sample has been found to use beta,beta-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed that penicillamine was derivatized to a phosphoramide adduct with the ADP moiety of NAD, whereas the nicotinamide-ribose group was released and hydrolyzed spontaneously to ribose and nicotinamide. The phosphoramide product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. The enzyme showed high substrate specificity to NAD (Km = 13.0 mM) and L-penicillamine (Km = 6.5 mM); other nucleotides such as NADP, NAD(P)H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with NAD as the first substrate to bind and ADP-L-penicillamine as the last product released. The novel NAD-dependent enzyme may catalyze the first step in penicillamine degradation in the strain of B. sphaericus.

Protective effect of adenylate deaminase (from Penicillium lanoso-viride) against acute infections in mice

We examined the effects of the immunomodulator-adenylate deaminase (E.C. 3.5.4.6) from Penicillium lanoso-viride on experimental mice infections. Prophylactic intraperitoneal administration of adenylate deaminase (ADA) increased survival time and numbers of survivors after infection with Salmonella typhimurium, Pseudomonas aeruginosa and influenza A (H3N2) virus. Protection against influenza virus after intranasal ADA application was also observed. The influence of ADA was time and dose dependent. The most pronounced protection was obtained by administration of 3 U ADA/mice 24 h prior to infection. ADA had no antibiotic effect against these bacterial strains. Protective effects of ADA were studied in immunosuppressed mice under different regimes of treatment including cyclosporin A and trypan blue. The results indicated, that the protective effect of ADA is of a complex nature and probably depends on both T-cell and macrophage components.

6-Substituted Derivatives of Carbovir: Anti-HIV Activity

Abstract A series of 6-alkoxy and 6-alkylamino carbovir derivatives were synthesized in order to evaluate prodrug approaches to increased bioavailability of the anti-HIV agent, carbovir. All of the compounds were active against HIV with the N-alkyl derivatives less active than the corresponding O-alkyl derivatives. The adenosine deaminase inhibitor, EHNA, had no effect on the anti-HIV activity of 6-propoxycarbovir, while the adenylic acid deaminase inhibitor, 2′-deoxycoformycin, significantly decreased antiviral activity. These observations suggest that the 6-alkoxycarbovirs are metabolized directly to the monophosphates and are subsequently converted to carbovir monophosphate via adenylic acid deaminase

Oxidative modulation and inactivation of rabbit cardiac adenylate deaminase.

Oxidative stress and adenine nucleotide catabolism occur concomitantly in several disease states, such as cardiac ischaemia-reperfusion, and may act as synergistic determinants of tissue injury. However, the mechanisms underlying this potential interaction remain ill-defined. We examined the influence of oxidative stress on the molecular, kinetic and regulatory properties of a ubiquitous AMP-catabolizing enzyme, adenylate deaminase (AMPD) (EC 3.5.4.6). To this intent, rabbit heart AMPD and an H2O2/ascorbate/iron oxidation system were employed. Enzyme exposure to the complete oxidation system acutely impaired its catalytic activity, lowered the Vmax. by 7-fold within 5 min, and rendered the enzyme unresponsive to nucleotide effectors. Irreversible AMPD inactivation resulted within about 15 min of oxidative insult and was not prevented by free-radical scavengers. Oxidative stress did not affect the molecular mass, tetrameric nature, Km, immunoreactivity or trypsinolytic pattern of the enzyme; nor did it induce carbonyl formation, Zn2+ release from the holoenzyme or net AMPD S-thiolation. This injury pattern is inconsistent with a radical-fragmentation mechanism as the basis for the oxidative AMPD inactivation observed. Rather, the sensitivity of the enzyme to both S-thiolation and thiol alkylation and the significant (3 of 9/mol of denatured enzyme) net loss of DTNB-reactive thiols on exposure to oxidant strongly implicate the conversion of essential thiol moieties into stable higher-oxidation states in the oxidative inactivation of cardiac AMPD. The altered thiol status of the enzyme on oxidative insult may prohibit a catalytically permissible conformation and, in so doing, increase AMP availability to 5'-nucleotidase in vivo.

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

Until now, the catabolism of adenosine(5′)triphospho(5′)adenosine (Ap 3A) and adenosine(5′)tetraphospho(5′)adenosine (Ap 4A) 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 (Ap nAs) undergo successive deamination leading, via an inosine(5′)oligophospho(5′)adenosine (Ip nA), to the corresponding inosine(5′)oligophospho(5′)inosine (Ip nI). 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; Ap 2A, Ap 3A, Ap 4A or Ap 5A. The K m values for these substrates are 20, 22, 32 and 39 μM, respectively, whereas the K m for 5′-AMP is 12 μM. 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 Ap 3A.

Mammalian-heart adenylate deaminase: cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody.

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (approximately 81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.

Age-related changes of AMP breakdown in chicken heart

The activity of adenylate deaminase, adenylate phosphatase and adenosine deaminase, as well as the endogenous content of adenine nucleotides, was examined in the heart of ageing chickens. In new-born (1-day-old) and young (20-day-old) chickens, AMP degradation in the heart seems to proceed preferentially through deamination, while in adult (1-year-old) through dephosphorylation. Compared with the adult heart, a 2-year-old one exhibits a decline of AMP catabolism. The total adenine nucleotide content and the concentration of ATP are higher in adult and aged chicken hearts, than in new-born and young ones. Adaptive mechanisms might occur in the heart of ageing chickens to ensure an adequate availability of adenine nucleotides.

The highly conserved Chinese hamster GNAI3 gene maps less than 60 kb from the AMPD2 gene and lacks the intronic U6 snRNA present in its human counterpart.

The identity of a gene coamplified with the adenylate deaminase 2 gene (AMPD2) in coformycin-resistant cells was determined by analysis of its genomic sequence. Sequence comparisons reveal a significant homology with the 3' terminal part of the gene encoding the alpha i3 subunit of Gi proteins from several species (GNAI3). Identification of the gene was confirmed by Western blot analysis of its products. A precise sequence comparison was performed with the human genomic sequence. It showed that conservation remains important in noncoding exons as well as in introns. However, sequences corresponding to combined U6 snRNA and E protein pseudogene, previously identified inside intron 7 of the human gene, were not found in the Chinese hamster gene. GNAI3 is mapped to a region of conserved linkage between human chromosome 1 (locus 1p13) and mouse chromosome 3 (at 48.4 cM). The Chinese hamster GNAI3 gene maps to chromosome 1 within a 120-kb fragment that also comprises the AMPD2 and GSTM genes.

Cardiac adenylate deaminase: molecular, kinetic and regulatory properties under phosphate-free conditions.

Adenylate deaminase (EC 3.5.4.6) may help to regulate the adenine nucleotide catabolism characteristic of such disease states as myocardial ischaemia. We report analysis of the molecular, kinetic and allosteric properties of rabbit heart adenylate deaminase when extracted and purified under phosphate-free conditions (i.e., with Hepes/KOH). The enzyme's subunit molecular mass (approximately 81 kDa), pI (6.5), substrate specificity for 5'-AMP, and activation by K+ were identical in the absence or presence of phosphate. At each chromatographic step during isolation without phosphate, cardiac adenylate deaminase showed a lower apparent activity as compared with the enzyme prepared with phosphate present. Kinetic constants for the phosphate-free rabbit heart adenylate deaminase preparation (Km 0.54 mM AMP; Vmax. 1.4 mumol/min per mg of protein) were approximately 10-fold lower than those of the enzyme isolated with phosphate. The same irreversible decrease in kinetic constants could be achieved by dialysing phosphate from the phosphate-containing enzyme preparation. The relationship between enzyme activity and substrate concentration was sigmoidal in the presence of phosphate, but hyperbolic in its absence. Cardiac adenylate deaminase under phosphate-free conditions was no longer allosterically activated by ATP and ADP, yet remained inhibitable by GTP. Enzyme inhibition by the transition-state mimic coformycin was not influenced by phosphate status. The phosphate-free preparation of rabbit heart adenylate deaminase was markedly labile and extremely susceptible to proteolysis by trypsin or chymotrypsin. The inactivation kinetics and fragmentation pattern in response to controlled proteolysis depended on whether the enzyme had been isolated with or without phosphate present, suggesting a conformational difference between the two enzyme preparations. These data constitute direct evidence that the absence of phosphate irreversibly converts cardiac adenylate deaminase into a pseudo-isoenzyme with distinct kinetic, regulatory and stability properties.