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PubMed1986

Protein properties of the subunits of ribonucleotide reductase and the specificity of the allosteric site(s).

Ribonucleotide reductase catalyzes the critical reaction in which the deoxyribonucleotides required for DNA replication are synthesized de novo. This enzyme consists of two non-identical protein subunits, both of which are required for enzymatic activity. These subunits consist of a non-heme iron and an effector-binding subunit. These subunits are not coordinately regulated as the cells pass from G1 to the S phase of the cell cycle. Studies carried out with the holoenzyme and the isolated subunits indicate that the effector-binding subunit is more susceptible to chymotrypsin and the sulfhydryl reagents, pCMB and NEM, than is the non-heme iron subunit. The non-heme iron subunit is more susceptible to trypsin than is the effector-binding subunit. The presence of ATP or dATP protects the effector-binding subunit from proteolysis by either trypsin or chymotrypsin. The loss of activity in the holoenzyme, as a result of proteolysis, parallels the loss of the particular subunit. These results demonstrate that the protein properties of the subunits are significantly different to account for the differential turnover. The binding of nucleotides to the effector-binding site(s), which in turn regulates ribonucleotide reductase activity, is very specific. Formycin 5'-triphosphate and etheno-ATP could not replace ATP in the CDP reductase reaction. 2',3'-DideoxyATP was 5-fold less active than dATP as a negative effector; etheno-dATP was not inhibitory. AraGTP and BuPdGTP could not replace dGTP as a positive effector of ADP reduction. BuPdGTP, but not araGTP, served as an inhibitor of CDP reduction. 2',3'-DideoxyTTP was much less active as either an activator of GDP reduction or an inhibitor of ADP reduction. These studies indicate that the binding to the allosteric sites is highly specific and suggest that the structural requirements for the binding of activators are different from the structural requirements for the binding of inhibitors.

Adenine Nucleotides↗

PubMed1993

Reduced purine accumulation is encoded on an amplified DNA in Leishmania mexicana amazonensis resistant to toxic nucleosides.

Nucleoside analogs are potential anti-Leishmania agents. To better understand how these compounds might lose their effectiveness, Leishmania were independently selected for resistance to inosine dialdehyde or tubercidin. Each of the resistant cells exhibited resistance to inosine dialdehyde and tubercidin as well as to formycin B and allopurinol ribonucleoside. Resistant cells had a greatly reduced capability of accumulating exogenous adenosine, guanosine, thymidine and guanine. This decreased ability to accumulate nucleosides and at least one nucleobase appeared to be due to reduced activity of a number of distinct purine transporters, as the differences between purine metabolizing enzymes were not sufficiently different to account for the decreased accumulation capability. The resistance to toxic nucleosides and the decreased ability to accumulate purines were due to the presence in the resistant cells of an extrachromosomal DNA approximately 55 kb in size. The extrachromosomal DNA was not detected in wild-type cells or revertants which have lost resistance to toxic nucleosides. Except for a 1.2-kb difference, the extrachromosomal DNA from both independently selected resistant cells appeared to be identical. The resistant cells contained 2-4 times as much DNA homologous to the extrachromosomal DNA as compared to wild type cells. When cloned into an E. coli/Leishmania shuttle vector, a portion of the amplified DNA had the ability to confer upon wild-type cells resistance to the toxic purine nucleoside analogs tubercidin and inosine dialdehyde. These transformed cells also exhibited a decreased ability to accumulate non-toxic purine nucleosides.

Animals↗

PubMed1986

Action of pyrazolopyrimidine derivatives on American Leishmania spp. promastigotes.

The capacity of 54 different pyrazolo-(3,4-d)- or -(4,3-d)-pyrimidine derivatives to inhibit American Leishmania promastigote multiplication was evaluated. Among pyrazolo-(3,4-d)-pyrimidines, eight derivatives showed leishmanistatic activity, 4-aminopyrazolo-(3,4-d)-pyrimidine (APP) being the most active, about eight-fold more than 4-hydroxy-pyrazolo-(3,4-d)-pyrimidine (HPP). 7-Hydroxy-3-beta-D-ribofuranosylpyrazolo-(4,3-d)-pyrimidine (FoB) was as active as 7-amino-3-beta-D-ribofuranosylpyrazolo-(4,3-d)-pyrimidine (FoA), a situation different to that found for pyrazolo-(3,4-d)-pyrimidines. Furthermore, different chemical modifications in formycin structure did not modify inhibitory effects. It can be concluded that regarding American Leishmania the chemical analogy to hypoxanthine or inosine of pyrazolo-(3,4-d)- and pyrazolo-(4,3-d)-pyrimidine, respectively, is not absolutely critical, as different modifications on the heterocyclic ring did not abolish the inhibitory activity of these compounds.

Animals↗

PubMed2006

Point mutations within the LdNT2 nucleoside transporter gene from Leishmania donovani confer drug resistance and transport deficiency.

Leishmania donovani, a protozoan parasite, expresses an unusual inosine/guanosine-specific transporter, LdNT2, the gene for which was cloned by functional rescue of a drug-resistant, LdNT2-deficient (FBD5) strain. In this investigation, we have uncovered and characterized the mutations within the LdNT2 open reading frame that are the basis for the drug-resistance and transport-incompetent phenotype of the FBD5 line. The FBD5 cells were shown to be compound heterozygotes in which both mutant ldnt2 alleles harbor discrete point mutations, each of which impaired transport function and conferred resistance to formycin B, the drug to which the clonal FBD5 line was selected. One of the mutant ldnt2 alleles encoded an S189L alteration in predicted transmembrane domain 5, while the second allele accommodated a null mutation at codon 376, which truncated the transporter just prior to transmembrane domain 8. In addition to the null transport phenotype, very little S189L ldnt2 mutant transporter targeted to the surface of the parasite. The bulk of the truncated ldnt2 appeared to be sequestered internally, possibly within the endoplasmic reticulum, but some of the truncated transporter seemed to be cell surface exposed. The ability to dissect mutations within a viable parasite offers LdNT2 as an attractive model for implementing a thorough forward genetic dissection of transporter function in a eukaryotic cell.

Animals↗

PubMed1991

A novel mechanism of cation/substrate cotransport: Na+/H+/adenosine cotransport in Vibrio parahaemolyticus.

Adenosine is actively transported with Na+ in Vibrio parahaemolyticus (Sakai, Y., Tsuda, M., Tsuchiya, T. (1987) Biochim, Biophys. Acta 893, 43-48). The proton conductor carbonylcyanide m-chlorophenylhydrazone, CCCP, strongly inhibited active transport of adenosine at pH 8.5 as well as at pH 7.0. This seemed peculiar because the driving force, an electrochemical potential of Na+, is established by the Na(+)-extruding respiratory chain at pH 8.5 in this organism, although it is established by the function of the Na+/H+ antiporter at pH 7.0. This suggested that H+ might be involved in the adenosine transport. We detected H+ uptake induced by adenosine influx in V. parahaemolyticus cells in the presence of Na+, but not in its absence, suggesting the occurrence of Na+/H+/adenosine cotransport. We isolated formycin A-resistant mutants which showed defective adenosine transport. The mutation resulted in simultaneous losses of Na+ uptake and H+ uptake induced by adenosine. In revertants from these mutants the Na+ uptake and H+ uptake were restored simultaneously. The frequencies of reversion were in the order of 10(-7), indicating that the mutations were single mutations; namely that Na+/adenosine cotransport and H+/adenosine cotransport took place via the same carrier. Thus, we conclude that adenosine is transported by the novel mechanism of Na+/H+/adenosine cotransport in V. parahaemolyticus.

Adenosine↗

PubMed1979

Cryptofluorescent analogs of cobalamin coenzymes: synthesis and characterization.

Upper-axial (beta-position) ligand analogs of the B12 coenzymes 5'-deoxy-5'-adenosylcob(III)alamin and methylcob(III)alamin have been synthesized by reaction of the 5'-chloro-5'-deoxy derivatives of fluorescent nucleosides (1,N6-ethenoadenosine, formycin, 2-amino-nebularine, and 2,6-diaminonebularine) and a fluorescent alkyl halide (dansylamidopropyl chloride) with cob(I)alamin. These analogs were nonfluorescent, but fluorescent products could be generated by photolysis or cyanolysis of the carbon-cobalt bonds. Under anaerobic and aerobic conditions, the major fluorescent photolysis products of 1,N6-ethenoadenosylcob(III)alamin were 1,N6-etheno-5',8-cyclic-5'-deoxyadenosine, and the 5'-aldehyde of 1,N6-ethenoadenosine, respectively. The cryptofluorescent property of these analogs was utilized to follow the kinetics of aerobic photolysis. First-order rate constants determined by this method were comparable to those obtained spectrophotometrically [via appearance of of aquacob(III)alamin]. Pseudo-first-order rate constants determined fluorometrically for the cyanolysis (at 25 degrees C) of 1,N6-ethenoadenosylcob(III)alamin, 2,6-diaminonebularinylcob(III)alamin, 2-aminonebularinylcob(III)alamin and formycinylcob(III)alamin were 5.8 X 10(-2), 2 X 10(-2), 1.8 X 10(-2), and 3 X 10(-5) min-1, respectively; values in good agreement were obtained spectrophotometrically (via appearance of dicyanocobalamin). Dansylamidopropylcob(III)alamin was stable in the presence of cyanide. The nucleoside alpha-ribazole is fluorescent in the free state but nonfluorescent when present as the lower axial (alpha-position) ligand in cobalamin coenzymes. Thus fluorescence of ligands in both the alpha- and beta-positions of cobalamins is quenched, probably as a result of intramolecular energy transfer between the ligands and the nonfluorescent corrinoid.

Aerobiosis↗

PubMed1994

The N-glycosidase mechanism of ribosome-inactivating proteins implied by crystal structures of alpha-momorcharin.

BACKGROUND: alpha-Momorcharin (alpha MMC) is a type I ribosome-inactivating protein. It inhibits protein synthesis by hydrolytically removing a specific adenine residue from a highly conserved, single-stranded loop of rRNA. RESULTS: Here we describe the determination and refinement of the crystal structures of alpha MMC in the native state and in complexes with the product, adenine, and a substrate analogue, formycin 5'-monophosphate (FMP) at high resolution. Both adenine and the base of FMP are tightly bound; the ribose of bound FMP adopts a strained, high-energy conformation, which may mimic the structure of the transition state. CONCLUSIONS: These structures indicate that residues Tyr70, Glu160 and Arg163 of alpha MMC are the most critical for catalysis. We propose that the strained conformation of the ribose in the target adenosine weakens the glycoside bond. Partial protonation mediated by Arg163 then facilitates N-glycoside bond cleavage, leading to the formation of an oxycarbonium ion intermediate which is stabilized by the negatively-charged Glu160. Tyr70 adopts subtly different conformations in the three structures implying that it may be important in substrate recognition and perhaps catalysis.

Amino Acid Sequence↗

PubMed1991

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects.

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1982

Synthesis of 9-(3,4-dioxopentyl)hypoxanthine, the first arginine-directed purine derivative: an irreversible inactivator for purine nucleoside phosphorylase.

The synthesis of two potential arginine-directed purine-based analogues, 6-chloro-9-(3,4-dioxopentyl)purine (6) and 9-(3,4-dioxopentyl)hypoxanthine (7), is reported. Compound 7 was extensively tested as a potential affinity label of purine nucleoside phosphorylase (EC 2.4.2.1) from human erythrocytes. Evidence that 7 reacted with the catalytic center of purine nucleoside phosphorylase includes the following: (1) time-dependent inactivation of the enzyme by 7 was observed; (2) a plot of the pseudo-first-order rate constant for inactivation of the enzyme vs. concentration of 7 was hyperbolic, characteristic of saturation phenomenon; (3) substrates (Pi, arsenate, inosine) and a competitive inhibitor (formycin B) protected the enzyme from inactivation by 7. Compound 7 was 25 times more effective in inhibiting purine nucleoside phosphorylase than butanedione. Evidence that 7 modified arginine(s) includes the following: (1) when the inactivation was performed in borate, both the rate and the extent of inactivation were enhanced compared to those of the controls run in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer; (2) dialysis of inactivator reversed the inactivation in Tris-HCl but not in borate buffer. All the above evidence combined with the previous demonstration [Jordan, F., & Wu, A. (1978) Arch. Biochem. Biophys. 190, 699-704] that butanedione modified only arginines in purine nucleoside phosphorylases and the results presented here demonstrating the similarities in the behavior of butanedione and 7 imply that compound 7 can be called an arginine-directed affinity label for purine nucleoside phosphorylase.

Hypoxanthines↗

PubMed1983

2',5'-Oligoadenylates and related 2',5'-oligonucleotide analogues. 1. Substrate specificity of the interferon-induced murine 2',5'-oligoadenylate synthetase and enzymatic synthesis of oligomers.

The substrate specificity of the interferon-induced mouse L-cell enzyme, 2',5'-oligoadenylate synthetase, was determined with a number of nucleoside 5'-triphosphate analogues. Selected nucleoside 5'-triphosphates were converted to 2',5'-oligonucleotides with the following order of efficiency for the nucleoside: 8-azaadenosine greater than adenosine = 2-chloroadenosine greater than sangivamycin greater than toyocamycin greater than formycin greater than 3-ribosyladenine greater than ribavirin greater than tubercidin greater than adenosine 1-oxide greater than 2-beta-D-ribofuranosylthiazole-4-carboxamide greater than inosine = 1,N6-ethenoadenosine greater than guanosine greater than 8-bromoadenosine = uridine greater than cytidine. Adenosine 5'-((beta, gamma-imidotriphosphate) did not seem to be a recognizable substrate since no detectable product resulted. Either the 2',5'-oligoadenylate synthetase is not as specific as had been previously thought, or there may be more than one 2',5'-oligonucleotide synthetase. The 2',5'-oligonucleotide analogue products in which the adenosine of ppp(A2'P5')nA was replaced by the various nucleoside analogues were separated by DEAE-cellulose column chromatography and the chain length and number of 5'-phosphate residues analyzed by a rapid, efficient high-performance liquid chromatographic (HPLC) system involving ion-pairing C18 reversed-phase column chromatography. Separation of the 5'-mono-, 5'-di-, and 5'-triphosphorylated forms of the 2',5'-oligonucleotide analogue dimers, trimers, tetramers, and pentamers was readily achieved by this useful HPLC system. No 5'-nonphosphorylated forms were detected for any of the 2',5'-oligonucleotide analogue products.

2',5'-Oligoadenylate Synthetase↗

PubMed1983

2',5'-Oligoadenylates and related 2',5'-oligonucleotide analogues. 2. Effect on cellular proliferation, protein synthesis, and endoribonuclease activity.

A number of the new enzymatically synthesized 2',5'-oligonucleotide trimers, namely, those containing the nucleosides 8-azaadenosine, toyocamycin, sangivamycin, formycin, 8-bromoadenosine, tubercidin, and guanosine, were found to inhibit protein synthesis and cellular proliferation after uptake into intact L and HeLa cells. 2',5'-Oligonucleotide trimers containing cytidine, inosine, uridine, and 1,N6-ethenoadenosine had some effect while those containing 2-chloroadenosine, 3-ribosyladenine, ribavirin, and 2-beta-D-ribofuranosylthiazole-4-carboxamide had no detectable effect on protein synthesis or cellular proliferation after uptake into L or HeLa cells. All of these 2',5'-oligonucleotide analogues inhibited protein synthesis in the in vitro rabbit reticulocyte lysate system except for the trimer containing ribavirin. Such nucleoside substitutions have further defined the substrate-specificity requirements for the endoribonuclease and/or the inhibitors for the 2',5'-phosphodiesterase. Most of the 2',5'-analogues were degraded in L-cell extracts so the endogenous nucleases are not very specific. The 2',5'-trimers containing tubercidin and 2-beta-D-ribofuranosylthiazole-4-carboxamide were quite stable in comparison to the 2',5'-A trimer. The inhibition of protein synthesis and cellular proliferation observed correlated well with the degradation of rRNA and polyadenylated mRNA observed after uptake of the 2',5'-analogues into intact L cells. The degradation of the polyadenylated mRNA appeared to be a more sensitive test than inhibition of cellular protein synthesis for determining biological activities of the 2',5'-oligonucleotide analogues.

Adenine Nucleotides↗

PubMed1981

Adenosine analogues as substrates and inhibitors of S-adenosylhomocysteine hydrolase.

In the reaction adenosine + L-homocysteine = S-adenosyl-L-homocysteine, catalyzed by S-adenosylhomo-cysteine hydrolase from beef liver (EC 3.3.1.1), 11 nucleosides are able to substitute for adenosine to generate their corresponding S-nucleosidylhomocysteine congeners: 3-deaza-adenosine, 2-aza-3-deazaadenosine, nebularine (purine ribonucleoside), formycin, N6-methyladenosine, 8-azaadenosine, adenosine N1-oxide, pyrazomycin, 8-aminoadenosine, inosine, and the carbocyclic analogue of adenosine [(+/-)-aristeromycin]. S-Adenosylhomocysteine hydrolase from lupin seeds is able to utilize all of these nucleosides except inosine to synthesize analogues of S-adenosylhomocysteine. There is no correlation between the ability of these nucleotides to function as substrates and their inhibitory potencies, except in the case of 3-deazaadenosine. The carbocyclic analogue of adenosine is the most potent inhibitor of S-adenosylhomocysteine hydrolase with a Ki of 5 X 10(-9) M. When incubated with 3T3-L1 fibroblasts, the carbocyclic analogue of adenosine caused a 20-fold increase in the cellular concentration of S-adenosyl-homocysteine. The cellular generation of S-2-aza-3-deaza-adenosylhomocysteine was observed when 3T3-L1 fibroblasts were incubated with 2-aza-3-deazaadenosine.

Adenosine↗

PubMed1980

Adenosine analogues as substrates and inhibitors of S-adenosylhomocysteine hydrolase in intact lymphocytes.

A number of adenosine analogues have been examined for their ability to interact with S-adenosyl-L-homocysteine (SAH) hydrolase in intact mouse lymphocytes. In the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, 3-deazaadenosine, 8-azaadenosine, formycin A, 2-aminoadenosine, 2-fluoroadenosine, N6-methyladenosine, N6-hydroxyadenosine, purine ribonucleoside and inosine were each metabilized to radioactive analogues of SAH when cells were labeled with either L-[2-3H]methionine or L-[35S]homocysteine. Tubercidin was shown to undergo metabolism both to S-[3H]tubercidinyl-L-methionine and to S-[3H]tubercidinyl-L-homocysteine in cells labeled with [2-3H]methionine. 9-beta-D-Arabinofuranosyladenine and 2'-deoxyadenosine caused marked elevations of [3H]SAH in cells preloaded with [2-3H]methionine but were not themselves metabolized detectably to SAH analogues. Adenine and 5'-deoxyadenosine also caused substantial elevations of [3H]SAH under these same conditions. Some of the adenosine analogues shown to be metabolized to SAH analogues also caused an elevation of SAH in the lymphocytes. These results indicate the potential of adenosine analogues to interfere with cellular methylation reactions due either to their inhibition of SAH hydrolase or to their metabolism, via this enzyme, to SAH analogues.

Adenosine↗

PubMed1998

A 1.4 A crystal structure for the hypoxanthine phosphoribosyltransferase of Trypanosoma cruzi.

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi, etiologic agent of Chagas' disease, was cocrystallized with the inosine analogue Formycin B (FmB) and the structure determined to 1.4 A resolution. This is the highest resolution structure yet reported for a phosphoribosyltransferase (PRT), and the asymmetric unit of the crystal contains a dimer of closely associated, nearly identical subunits. A conserved nonproline cis peptide in one active-site loop exposes the main-chain nitrogen to the enzyme active site, while the adjacent lysine side chain interacts with the other subunit of the dimer, thereby providing a possible mechanism for communication between the subunits and their active sites. The three-dimensional coordinates for the invariant Ser103-Tyr104 dipeptide are reported here for the first time. These are the only highly conserved residues in a second active-site loop, termed the long flexible loop, which is predicted to close over the active site of HPRTs to protect a labile transition state [Eads et al. (1994) Cell 78, 325-334]. This structure represents a major step forward in efforts to design/discover potent selective inhibitors of the HPRT of T. cruzi.

Animals↗

PubMed1972

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17 .

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.

Adenine Nucleotides↗

PubMed1996

Nucleoside uptake in rat liver parenchymal cells.

Rat liver parenchymal cells express Na(+)-dependent and Na(+)- independent nucleoside transport activity. The Na(+)-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227-233]. This transport activity shows apparent K(m) values for uridine in the range 8-13 microM and a Vmax of 246 pmol of uridine per 3 min per 10(5) cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na(+)-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na(+)-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na(+)-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity that is inhibited by high concentrations of thymidine is similar to the guanosine-resistant fraction. These observations are consistent with the presence of at least two Na(+)-dependent transport systems. Na(+)-dependent uridine uptake is sensitive to N-ethylmaleimide treatment, but Na(+)-independent transport is not. Nitrobenzylthioinosine (NBTI) stimulates Na(+)-dependent uridine uptake. The NBTI effect involves a change in Vmax, it is rapid, dose-dependent, does not need preincubation and can be abolished by depleting the Na+ transmembrane electrochemical gradient. Na(+)-independent uridine transport seems to be insensitive to NBTI. Under the same experimental conditions, NBTI effectively blocks most of the Na(+)-independent uridine uptake in hepatoma cells. Thus the stimulatory effect of NBTI on the concentrative nucleoside transporter of liver parenchymal cells cannot be explained by inhibition of nucleoside efflux.

Affinity Labels↗

PubMed1978

Interaction between actomyosin and 8-substituted ATP analogs.

Various 8-substituted ATP analogs were synthesized, and their reactions with myosin and actomyosin were studied. The nucleoside triphosphates (NTPs) with an amino group at the 6 position and hydrogen at the 8 position, and formycin 5'-triphosphate (FTP) were hydrolyzed by myosin very slowly in the presence of Mg2+ and rapidly in the presence of EDTA and K+. In contrast, NTPs with substitution of the 8 position, other than FTP, were readily hydrolyzed by myosin in the presence of Mg2+ but were hardly hydolyzed in the presence of EDTA and K+. The Michaelis constant (Km) for hydrolysis of 8-substituted NTP by heavy meromyosin was much larger than the dissociation constant (Kfl) for binding of heavy meromyosin with NTP estimated from the change in tryptophan fluorescence. All the NTPs with no substitution at the 8 position, and FTP, caused an initial Pi burst, actin activation of myosin NTPase, superprecipitation of actomyosin, and myofibrillar contraction. On the other hand, all the 8-substituted NTPs in three possible conformations did not cause these phenomena, regardless of the conformation. These results were discussed in relation to the hindrance of rotation about the glycosidic bond accompanying an 8 substitution.

Actomyosin↗

PubMed2001

Three-dimensional structure of a hyperthermophilic 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus.

The structure of 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) has been determined alone, as ternary complexes with sulfate plus substrates 5'-deoxy-5'-methylthioadenosine, adenosine, or guanosine, or with the noncleavable substrate analog Formycin B and as binary complexes with phosphate or sulfate alone. The structure of unliganded SsMTAP was refined at 2.5-A resolution and the structures of the complexes were refined at resolutions ranging from 1.6 to 2.0 A. SsMTAP is unusual both for its broad substrate specificity and for its extreme thermal stability. The hexameric structure of SsMTAP is similar to that of purine-nucleoside phosphorylase (PNP) from Escherichia coli, however, only SsMTAP accepts 5'-deoxy-5'-methylthioadenosine as a substrate. The active site of SsMTAP is similar to that of E. coli PNP with 13 of 18 nearest residues being identical. The main differences are at Thr(89), which corresponds to serine in E. coli PNP, and Glu(163), which corresponds to proline in E. coli PNP. In addition, a water molecule is found near the purine N-7 position in the guanosine complex of SsMTAP. Thr(89) is near the 5'-position of the nucleoside and may account for the ability of SsMTAP to accept either hydrophobic or hydrophilic substituents in that position. Unlike E. coli PNP, the structures of SsMTAP reveal a substrate-induced conformational change involving Glu(163). This residue is located at the interface between subunits and swings in toward the active site upon nucleoside binding. The high-resolution structures of SsMTAP suggest that the transition state is stabilized in different ways for 6-amino versus 6-oxo substrates. SsMTAP has optimal activity at 120 degrees C and retains full activity after 2 h at 100 degrees C. Examination of the three-dimensional structure of SsMTAP suggests that unlike most thermophilic enzymes, disulfide linkages play a key in role in its thermal stability.

Binding Sites↗