Adenine Analogue Research Articles

BMS-200475, a novel carbocyclic 2′-deoxyguanosine analog with potent and selective anti-hepatitis B virus activity in vitro

BMS-200475, a novel carbocyclic analog of 2′-deoxyguanosine, is a potent inhibitor of hepatitis B virus in vitro (ED 50 = 3 nM) with relatively low cytotoxicity (CC 50 = 21–120 μM). A practical 10-step asymmetric synthesis was developed affording BMS-200475 in 18% overall chemical yield and >99% optical purity. The enantiomer of BMS-200475 as well as the adenine, thymine, and iodouracil analogs are much less active.

Enzymatic formation of potential anticancer and antiviral inosine analogues.

Theoretically, inosine analogues should act as effective inhibitors of tumor cell proliferation and viral replication. To acquire a broad spectrum of new candidate inosine analogues, a rapid, facile, quantitative and stereoselective method for deaminating potential antitumor and antiviral adenine analogues previously synthesized in our laboratory was developed. A novel 5'-adenylic acid deaminase, with relaxed substrate requirements, from Aspergillus species was utilized to deaminate four hexofuranosyladenine nucleosides and five adenine nucleoside dialdehydes to their corresponding inosine analogues. The fastest rates of deamination for the hexofuranosyl nucleosides were for the compounds where the vicinal hydroxyl groups on the sugars are oriented in the erythro configuration. For rapid deamination of the adenine nucleoside dialdehydes, the R configuration at the proximal carbon atom is preferred, while the nature of the group on the distal carbon atom has no significant effect on the rate or extent of deamination.

Chiral pyrrolo[2,3-d]pyrimidine and pyrimido[4,5-b]indole derivatives: structure-activity relationships of potent, highly stereoselective A1-adenosine receptor antagonists.

A series of 33 novel, mostly chiral pyrrolo[2,3-d]pyrimidine and pyrimido[4,5-b]indole derivatives has been synthesized and investigated in radioligand binding assays at the high-affinity adenosine receptor (AR) subtypes A1 and A2a. The compounds can be envisaged as adenine and hypoxanthine analogs lacking the nitrogen in the 7-position (7-deazaadenines and 7-deazahypoxanthines). 7-Deazaadenines were much more potent than 7-deazahypoxanthines at AR with A1AR affinities in the low-nanomolar range, extraordinarily high selectivity for the rat brain A1AR versus the A2aAR (several thousandfold), and high stereoselectivity (up to 96-fold). Pyrimido[4,5-b]indoles were more potent A1AR antagonists compared to pyrrolo[2,3-d]pyrimidines. Compound 34a (APEPI) is one of the most potent and most selective nonxanthine A1AR antagonists known to date (Ki = 2.8 nM, > 2000-fold A1-selective). A new class of very potent A1AR antagonists has been identified, namely, 2-phenyl-7-deazaadenines bearing a substituent at the exocyclic amino group (N4-substituted pyrrolo[2,3-d]pyrimidines). (R)-N- (1-Phenylethyl)-4-amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin e (DPEAP, 17a) showed a Ki value of 6.7 nM at A1AR and > 4000-fold A1 selectivity. Different binding modes are postulated for the N4-substituted 4-aminopyrrolo[2,3-d]pyrimidines (e.g., 17a) and the 7-substituted derivatives (e.g., 1a), based on a comparison of steric, electronic, and hydrophobic properties of the two classes of compounds. Water solubility and lipophilicity have been determined for selected compounds. 4-Amino-5,6-dimethyl-2-(3-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidine (4a) showed the highest water solubility/A1AR affinity ratio of 368 in the present series, over 2000-fold A1 selectivity, and 64-fold stereoselectivity (R > S). Therefore, 4a should be an interesting compound for in vivo evaluation.

The specific binding of Escherichia coli integration host factor involves both major and minor grooves of DNA.

The integration host factor (IHF) of Escherichia coli is a small, sequence-specific DNA-binding protein. The specific and nonspecific binding constants of IHF were estimated by gel-retardation assays. The equilibrium association constant of IHF for the H' site in lambda attP is 6.8 x 10(8) M-1 (Kd = 1.5 nM), and the nonspecific binding constant is 5.8 x 10(5) M-1 (Kd = 1.7 microM), giving a selectivity of approximately 1,000-fold for a specific site over random sequences. To study the molecular determinants specifying IHF binding, we used a series of 41 oligonucleotides containing adenine analogues that modified the surfaces of the major and minor grooves of the DNA. Many of the analogue substitutions within the previously defined consensus region caused decreased binding. Replacement with various analogues outside the consensus domain had little effect. Quantifying the binding constants for those sites with reduced affinities indicated an interaction with the minor groove within the consensus sequence. The binding constants of sites with 2-aminopurine and an inosine substitution within the same region suggest that IHF may also interact with the major groove. Thus, the specific interaction of IHF with its H' site likely involves interactions with both the minor and major grooves of the DNA.

Induction of G2 Arrest and Gene Expression by 2-Aminopurine in Human U937 Promonocyte-Macrophage Cells

The adenine analogue, 2-aminopurine (2-AP), induces G2 arrest in the human promonocyte-macrophage cell line, U937. The arrest is reversible and cells enter mitosis to resume normal logarithmic growth upon removal of the drug. These physiological changes are accompanied by markedly stimulated expression of eukaryotic gene constructs stably integrated in the chromosomes or introduced into the cells by transient transfection. Induction by 2-AP has two components: one involves increased transcription of the introduced genes as shown by run-on transcription experiments. The other involves markedly increased mRNA half-life that affects mRNA transcribed from transiently transfected DNA but apparently does not affect mRNA transcribed from the same chromosomally integrated sequences. Together, these two components could account for the 100- to 1000-fold inductions observed with various transfected gene constructs reported here and elsewhere. Maximum induction by 2-AP is promiscuous with respect to eukaryotic promoter origins or sequences, but appears to require a minimum of two such promoter elements. Thus, G2 cell cycle arrest induced by 2-AP is also associated with transcriptional and post-transcriptional alterations in gene expression. The data also suggest that transiently transfected DNAs may enter spatial or biochemical compartments of the nucleus that are different from those of normal genes in their native locations. These differences may affect the abundance and fate of the transcribed mRNA and, in some circumstances, introduce serious discordances into studies of gene regulation.

Pharmacological analysis of the interaction between purinoceptor agonists and antagonists in the guinea-pig taenia caecum.

1. In the absence of adenosine uptake inhibition, adenosine produced a concentration-dependent (threshold 30 microM) relaxation of the 5-methylfurmethide pre-contracted guinea-pig taenia caecum. The relaxation was not blocked by 8-phenyltheophylline (8-PT, 3 microM) or 1,3-dipropyl, 8-cyclopentylxanthine (DPCPX, 30 microM). 2. In the presence of the adenosine uptake inhibitor, dipyridamole (Dip, 3 microM), a biphasic adenosine concentration-effect curve was obtained (threshold 0.3 microM). The time course of the responses to adenosine in the absence of Dip was similar to that of the second phase responses in the presence of Dip and occurred over the same adenosine concentration-range. 5'-(N-ethyl) carboxamido-adenosine (NECA) concentration-effect curves (in the absence of Dip) were also biphasic. Only the first phases of the concentration-effect curves obtained with NECA and adenosine (plus Dip) were inhibited by 8-PT. The pA2 values for 8-PT of 6.7 and 7.0 versus adenosine and NECA, respectively, were consistent with actions at P1-purinoceptors. There was a trend towards an increase in the upper asymptote of the first phase of the NECA curve in the presence of increasing concentrations of 8-PT. The A1-purinoceptor selective antagonist, DPCPX, also blocked only the first phase of the NECA concentration-effect curve and produced a significant increase in the upper asymptote. The pA2 value (6.8) obtained was consistent with activation of A2-subtype P1-purinoceptors by the low concentrations of NECA. 3. There was no correlation between A1-purinoceptor affinity and the propensity to cause the increase in the upper asymptote of the first phase of the NECA concentration-effect curves amongst a series of 9-methyl adenine analogues, suggesting that the amplification was not due to inhibition of an underlying A1-purinoceptor-mediated contractile response.4. DPCPX (10 microM) produced a significant increase in the upper asymptote of the NECA concentration effect curve, but had no effect on isoprenaline curves whereas the phosphodiesterase inhibitor Ro20-1724 (30 microM) produced a significant increase in the upper asymptote of both NECA and isoprenaline concentration-effect curves. Therefore the amplification of the first phase responses by DPCPX did not appear to be due to phosphodiesterase inhibition.5. It was not possible to conclude whether second phase responses to adenosine and NECA were mediated by intracellular or extracellular sites of action. However, if intracellular sites of action were involved then adenosine did not apparently gain access by the Dip-sensitive uptake system.

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.

Increased stability of nucleic acids containing 7-deaza-guanosine and 7-deaza-adenosine may enable rapid DNA sequencing by matrix-assisted laser desorption mass spectrometry.

The use of matrix-assisted laser desorption mass spectrometry (MALDI-MS) has been suggested as an ultrafast readout of Sanger DNA sequencing ladders in a manner analogous to that used with sequencing gels. Currently, a serious limitation of MALDI-MS for the analysis of DNA results from the tendency for oligonucleotides to undergo facile fragmentation in the gas phase. The present study was undertaken to gain an understanding of the influence of various chemical structural features of purine bases on the stability of oligodeoxynucleotide ions produced by MALDI. The study focused on the stability of model compounds of the type d(TTTTTTTTTTXTTTTTTTTT TTTT), where T designates deoxythymidine and X a purine-containing 2'-deoxynucleotide. A variety of different purine derivatives were chosen as the base in the nucleotide X. The mass spectra of the model compounds containing 7-deaza analogues of guanine and adenine reveal a significantly increased stability compared to the 7-aza analogues under the conditions of MALDI-MS. The previously reported incorporation of the 7-deaza-2'-deoxy-adenosine triphosphate and the 7-deaza-2'-deoxy-guanosine triphosphate into DNA by polymerases suggests their use in a Sanger dideoxy sequencing experiment. The dideoxy termination products with the 7-deaza-purines instead of the 7-aza-purines might be sufficiently stable to allow separation and detection of the sequencing ladder by MALDI-MS. Thus, an ultrafast (seconds) read-out of DNA sequence may become feasible.

Isolation and characterization of mutations in the mouse APRT gene that encode functional enzymes with resistance to toxic adenine analogs.

Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) is a purine salvage enzyme that catalyses the reaction between adenine and 5-phosphoribosylpyrophosphate (PRPP) to form AMP. Expression of the aprt gene is not required for cell survival in culture, therefore, it may be selected against by growing cells in the presence of toxic adenine analogs such as 2,6-diaminopurine (DAP). In general, DAP-resistant cell lines do not exhibit significant levels of APRT activity (Taylor and Sahota, 1989).

Melting of a DNA helix terminus within the active site of a DNA polymerase.

Accurate synthesis of DNA by polymerase is due in part to the selective removal of misincorporated nucleotides by a 3'-5' exonuclease activity (proofreading). Proofreading by an exonuclease domain containing a single-stranded DNA binding site may involve local melting of a duplex DNA substrate. Here we use time-resolved fluorescence spectroscopy to analyze the local melting of a DNA duplex terminus induced by the Klenow fragment of DNA polymerase I. Four oligodeoxynucleotide primer/templates were prepared, each containing the fluorescent adenine analog 2-aminopurine (A*) at the primer 3' terminus, and one of the common DNA bases opposite the A* residue. Fluorescence decays of the duplex DNAs and the single primer oligonucleotide were jointly analyzed using global analysis procedures. Four lifetime components were resolved in the duplex DNAs, representing distinct conformational states of the terminal A* residue: paired A* bases, partially stacked A* bases, and extended A* bases. The variation of the apparent fraction of paired A* bases with temperature was in accord with optical melting data, and the extent of base pairing observed in each duplex was consistent with the base-pairing preferences of A* established in other studies. These results establish that the fluorescence decay characteristics of A* can be used to examine base-pairing interactions at a DNA duplex terminus. Since the fluorescence of A* can be observed without interference from protein amino acid residues, unlike existing methods for monitoring DNA melting transitions, this method was used to examine the extent to which Klenow fragment could induce fraying at each duplex terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole with the (Ca 2+ + Mg 2+) -ATPase protein of sarcoplasmic reticulum at low temperature

Modification of the (Ca 2+ + Mg 2+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum (SR) with 7-chloro-4-nitrobenzo-2-oxa 1, 3-diazole, NBD-Cl, at 4°C for 5 min caused a 63% loss of the Ca 2+-dependent ATPase activity when 1 mol of the adenine analog was incorporated per 10 5 g of protein. At 25°C, above the lipid phase transition, the extent of labeling was 3-fold higher although the Ca 2+-ATPase activity was inhibited to the saine extent. MgATP protected the ATPase activity at 4°C and 25°C but there was little change in the extent of labeling at 4°C suggesting that changes in the fluidity of the lipid moiety made different sites on the ATPase protein accessible to the reagent. At 4°C, addition of sodium deoxycholate enhanced the inactivation (6% ATPase activity remained) but the labeling of the SR-ATPase protein did not increase significantly. Incubation with MgATP prior to solubilization with deoxycholate resulted in the protection of the Ca 2+-ATPaseactivity and only a small decrease in the labeling occurred. At 25°C, a similar pattern was found with deoxycholate but the loss of ATPase activity was less dramatic and the extent of labeling by NBD-Cl was greater than that at 4°C. MgATP induced changes in the conformation of the ATPase protein protecting essential cysteine residues while shifting the reaction of NBD-Cl with the ATPase protein to non-essential sites in the absence or presence of deoxycholate. An analysis of tryptic digests of the NBD-ATPase protein showed that MgATP shifted the labeling from the A 2 subfragment to the A 1 subfragment in the absence of deoxycholate and from the A 1 subfragment to the A 2 subfragment in the presence of deoxycholate. The reagent, NBD-Cl, can distinguish between different temperature dependent conformational states of the ATPase protein.

Synthesis of 1‐(8‐phosphonomethoxy‐3,6‐dioxaoctyl)pyrimidines and purines, a novel series of acyclonucleotide analogues

AbstractA convenient synthesis of 1‐(8‐phosphonomethoxy‐3,6‐dioxaoctyl) uracil, cytosine, adenine, and guanine nucleotide analogues 26, 28, 30, and 32 by Mitsunobu coupling of the nucleobases with suitably functionalized alcohol derivative 24 is described. The antiviral activity of this series of compounds against herpes simplex virus (HSV) types 1 and 2 and human cytomegalovirus is reported.

A fluorescence-based assay for monitoring helicase activity.

A continuous fluorescence-based assay is described for measuring helicase-mediated unwinding of duplex DNA. The assay utilizes an oligonucleotide substrate containing the fluorescent adenine analog, 2-aminopurine, at regular intervals. 2-Aminopurine forms a Watson-Crick-type base pair with thymine and does not distort normal B-form DNA. Fluorescence of the 2-aminopurines within this oligonucleotide is quenched 2-fold upon its hybridization to a complementary strand. Unwinding of this substrate by the T4 dda helicase restores the fluorescence of the 2-aminopurines and is easily followed using stopped-flow or steady-state fluorescence spectroscopy. The flourescence-based assay provides rate data comparable to that obtained from conventional discontinuous assays using labeled substrates and additionally furnishes a means for following a single turnover. This assay should prove useful for defining the mechanism by which helicases unwind duplex DNA.

Additional evidence for the exceptional mechanism of the acid-catalysed hydrolysis of 4-oxopyrimidine nucleosides: hydrolysis of 1-(1-alkoxyalkyl)uracils, seconucleosides, 3′-C-alkyl nucleosides and nucleoside 3′,5′-cyclic monophosphates

The rate constants for the acid-catalysed hydrolysis of 1-(1 -alkoxyethyl)uracils and 1-alkoxymethyluracils have been determined. With both series of compounds, the hydrolysis rate is rather insensitive to the polar nature of the alkoxy group, in striking contrast with the hydrolysis of the corresponding analogues of adenine and cytosine nucleosides, which react via rate-limiting formation of an oxocarbenium ion intermediate. Furthermore, it has been shown that the 3′,5′-cyclic monophosphates of thymidine and uridine undergo the hydrolysis of the N-glycosidic bond 760 and 260 times as fast as their parent nucleosides, while the cyclic monophosphates of 2′-deoxyadenosine and adenosine are depurinated much more slowly than the corresponding nucleosides. On this basis it is suggested that 4-oxopyrimidine nucleosides are hydrolysed by opening of the sugar ring. To obtain further evidence for this exceptional mechanism, comparative kinetic measurements with some seco- and 3′-C-alkyl nucleosides of uracil and adenine have been carried out.

アデノシン発酵の工業的確立（第２報） Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓから誘導したアデニンアナログ耐性株によるアデノシン生産

A mutant resistant to adenine analogs was derived from an adenosine producer, Bacillus subtilis A 3-5, which had xanthine auxotrophy, 8-azaguanine resistance, and sulfaguanidine resistance. Selection during breeding was done by screening for resistance to 2-thioadenine sulfate (0.5mg/ml), resistance to 8-azaadenine (1mg/ml), and resistance to 6-methylaminopurine (1mg/ml), in that order. The strain obtained, No.43-35, produced more adenosine than the parent strain. For increased production of adenosine, the effects of the cultural conditions were examined. Strain No.43-35 produced 24 g/l adenosine from 130 g/l glucose in a glass jar fermenter with a working volume of 300 ml at the best cultural conditions found.

Nephrotoxicity induced by adenine and its analogs: relationship between structure and renal injury.

Twenty-four adenine analogs were administered to mice and the relationship between the structure of analogs and the occurrence of renal injury was examined. Plasma urea nitrogen (UN) and creatinine levels were measured 24 h after oral administration of analogs. Both levels increased in the adenine-, 8-azaadenine-, isoguanine-, or 6-dimethyl aminopurine (6-DMAP)-administered group, but did not increase in the other analog groups. From light microscopy, the damages of tubuli, mainly of proximal tubuli, were observed in the kidneys of these four groups. The common property of these compounds is the strong basicity of nitrogen which binds the 6-position of the purine ring. Furthermore, UN and creatinine increased time-dependently with intravenous administration of isoguanine. When adenine was intravenously administered, UN slightly increased at 1 h, but creatinine was unchanged. No changes were observed in the 6-DMAP- or 8-azaadenine-administered group. The basicity of nitrogen which binds to the 6-position of the purine ring is thus considered to be related to the occurrence of renal injury with oral administration, and isoguanine has high affinity with the kidney.

Identification of the adenine binding site in the ricin toxin A‐chain by fluorescence, CD, and electron spin resonance spectroscopy

AbstractCD, electron spin resonance, and fluorescence spectroscopy have been utilized to study the adenine binding site of ricin and its toxic A‐subunit. At acidic (4.5) and physiological (7.3) pH, adenine or a spin‐labeled analogue of adenine, N6‐(2,2,6,6‐tetramethyl‐1‐oxypiperidin‐4‐yl) adenine, alters the near uv CD spectra of the ricin A‐chain as well as intact ricin, whereas the far uv CD spectra of all proteins remain unchanged. Electron spin resonance data show that the adenine spin‐labeled analogue interacts strongly with the A‐chain both at pH 4.5 and 7.3, but no or very weak binding is observed for the intact ricin or the isolated B‐chain. The adenine spin label gets highly immobilized (2AII = 65.5G) by the A‐chain. The apparent dissociation constant Kd for the toxic A‐chain ligand complex is 1.55 × 10−4 M and 5.6 × 10−5 M at pH 7.3 and 4.5, respectively. Fluorescence intensity of ricin A‐chain bound 1,8‐anilinonaphthalenesulfonic acid (ANS) decreases by ∼55% at pH 4.5 with the addition of the spin‐labeled analogue of adenine, implying that both the ANS and adenine spin label (ADSL) bind to the hydrophobic domain of the A‐chain. Fluorescence of the only intrinsic tryptophan probe of the A‐chain is also efficiently quenched by ADSL, indicating that the tryptophan residue and the hydrophobic adenine binding site are closely located. All spectroscopic measurements indicate that adenine or its spin‐labeled analogue has a single binding site adjacent to the TRP211 residue in the A‐chain. Expansion of the A‐chain globule and subsequent exposure of the hydrophobic binding site seem to be responsible for the increased binding of adenine at pH 4.5. © 1993 John Wiley & Sons, Inc.

Synthesis and biological evaluation of nucleosides containing 8‐amino‐imidazo[1,2‐α]pyrazine as an isosteric replacement for adenine

AbstractA number of novel C‐nucleosides related to purine derivatives are described in which the purine moiety has been replaced by the isosteric heterocycle, 8‐aminoimidazo[1,2‐α]pyrazine. The nucleosides prepared include the ribo, 3′‐deoxy, 2′,3′‐dideoxy, and 2′,3′‐unsaturated derivatives. These C‐nucleosides represent derivatives containing acid stable glycosyl bonds and they can be considered as analogs of adenine‐ or 3‐deazaade‐nine‐containing nucleosides. Preparation of the parent ribonucleoside was accomplished by reaction of the C‐l functionalized sugar, (2ξ)‐1‐amino‐3,6‐anhydro‐l‐deoxy‐4,5‐O‐isopropylidene‐7‐O‐trityl‐D‐allo‐heptitol with 2,3‐dichloropyrazine, followed by ring closure to the 8‐chloroimidazo[1,2‐α]pyrazine nucleoside, conversion to the 8‐amino derivative and deblocking. A single crystal X‐ray structure of the parent 8‐amino‐3‐(β‐D‐ribofuranosyl)imidazo[1,2‐α]pyrazine is described and the conformation compared to that of formycin. The sugar‐modified analogs were prepared by subsequent functional group manipulations on the sugar moiety. Biological evaluation against HIV in H9 T‐lymphoid cell culture showed the nucleosides to be devoid of significant antiviral activity compared to DDA. The 3‐deazaadenosine analog also demonstrated weak suppression of mouse splenic NK activity toward YAC cells (mouse lymphoma cell targets). The imidazo[1,2‐α]pyrazine analog of 3‐deazaadenosine showed antiinflammatory activity in vivo in the rat pleurisy carrageenan model in the same range with 3‐deazaadenosine.

Phosphorylation and nucleotide-dependent dephosphorylation of hepatic polypeptides related to the plasma cell differentiation antigen PC-1

A glycoprotein fraction was isolated from rat liver membranes by affinity chromatography on immobilized wheat-germ lectin. Incubation of this fraction with MgATP or MgGTP resulted in a sequential phosphorylation and dephosphorylation of a complex of three polypeptides (118, 128 and 197 kDa on SDS/PAGE) with N-linked sialyloligosaccharides. Each polypeptide was recognized by polyclonal antibodies against recombinant plasma cell differentiation antigen PC-1. The relationship of the 118 kDa and 128 kDa polypeptides with PC-1 was confirmed by observations that they are linked by disulphide bonds into a larger protein, and that they are exclusively phosphorylated on Thr residues. Phosphorylation of p118, p128 and p197 only occurred after a lag period (up to 90 min at 30 degrees C), which lasted until most of the ATP had been converted to adenosine and Pi, with ADP and AMP as intermediate products. The length of the latency period increased with the concentration of initially added ATP (5-1000 microM) and could be prolonged by a second addition of similar concentrations of ATP, ADP, AMP and various nucleotide analogues. Most potent were the alpha beta-methylene derivatives of ADP and ATP. Adenosine was poorly effective. AMP, ADP, and perhaps ATP, emerge as the direct determinants of the latency. After further purification of the lectin-purified membrane fraction on anion-exchange and molecular-sieve columns, the complex of p118, p128 and p197 was still capable of autophosphorylation and dephosphorylation. The dephosphorylation was not affected by classical inhibitors (NaF, okadaic acid, EDTA, EGTA, phenylalanine). It was stimulated about 20-fold by various adenine nucleotides and analogues, with the same order of efficiency as noted for the induction of the latency. A similar stimulation of dephosphorylation was caused by 0.5 mM Na3VO4, which also prevented the phosphorylation of the three polypeptides. The likely explanation for the latency that precedes the phosphorylation of the membrane proteins is that the action of a protein kinase is initially offset by nucleotide-stimulated dephosphorylation.

The effect of 4-aminopyrazolo(3,4-d) pyrimidine on mouse plasma alpha-amylase activity.

A single dose of 4-aminopyrazolo(3,4-d) pyrimidine (4APP), an adenine analog, was orally administered at various concentrations of groups of mice and after 24 h, the plasma-alpha-amylase activity and cholesterol levels had significantly decreased in a dose-dependent manner. There was no change, however, in pancreatic superoxide dismutase activity or the lipid peroxide level. Histologically, zymogen degranulation was found after a high oral dose of 4APP (175 mg/kg). The same decrease in alpha-amylase activity occurred in mice that had received ip administration of 4APP, and in mice that were pretreated with dimethyl sulfoxide. Based on these results, it appears that 4APP decreases plasma alpha-amylase activity dose-dependently, possibly by pancreatic injury, and that free radicals play no part in the observed changes.