Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography

A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N⁶-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25–1.0 nmol/g wet weight for renal tissue and 0.25–0.5 μM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1±2.6% and 102.5±4.0% for renal tissue and urine, respectively (mean±S.D., n=3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64±2.2, 0.67±0.18 and 46.2±1.9 nmol/g wet weight, respectively (mean±S.D., n=6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low μM range and are negatively correlated with urine flow-rate.     

Myocardial S-adenosylhomocysteine hydrolase is important for adenosine production during normoxia

The coronary vasodilator adenosine can be formed in the heart by breakdown of AMP or S-adenosylhomocysteine (SAdoHcy). The purpose of this study was to get insight into the relative importance of these routes of adenosine formation in both the normoxic and the ischemic heart. A novel HPLC method was used to determine myocardial adenosine and SAdoHcy. Accumulation of SAdoHcy was induced in isolated rat hearts by perfusion with L-homocysteine thiolactone or L-homocysteine. The release of adenosine, inosine, hypoxanthine, xanthine and uric acid was determined. Additional in vitro experiments were performed to determine the kinetic parameters of S-adenosylhomocysteine hydrolase. During normoxia the thiolactone caused a concentration-dependent increase in SAdoHcy. At 2000 microM of the thiolactone an SAdoHcy accumulation of 0.49 nmol/min per g wet weight was found during normoxia. L-Homocysteine (200 microM) caused an increase of 0.37 and 4.17 nmol SAdoHcy/min per g wet weight during normoxia and ischemia, respectively. The adenosine concentration in ischemic hearts was significantly lower when homocysteine was infused (6.2 vs. 11.5 nmol/g; P less than 0.05). Purine release was increased 4-fold during ischemia. The Km for hydrolysis of SAdoHcy was about 12 microM. At in vitro conditions favoring near-maximal SAdoHcy synthesis (72 microM adenosine, 1.8 mM homocysteine), the synthesis rate in homogenates was 10 nmol/min per g wet weight. From the combined in vitro and perfusion studies, we conclude that S-adenosylhomocysteine hydrolase can contribute significantly to adenosine production in normoxic rat heart, but not during ischemia.     

The transmethylation pathway as a source for adenosine in the isolated guinea-pig heart.

In order to quantify adenosine production from the transmethylation pathway [S-adenosylmethionine (AdoMet)----S-adenosylhomocysteine (AdoHcy) in equilibrium adenosine + L-homocysteine] in the isolated guinea-pig heart under basal conditions (normoxic perfusion with 95% O2) and during elevated adenosine production (hypoxic perfusion with 30% O2), two methods were used. (1) Hearts were perfused with normoxic medium containing [2,5,8-3H]adenosine (5 microM) and L-homocysteine thiolactone (0.1 mM), which brings about net AdoHcy synthesis via reversal of the AdoHcy hydrolase reaction and labels the intracellular pool of AdoHcy. From the decrease in AdoHcy pool size and specific radioactivity of AdoHcy in the post-labelling period, the rate of transmethylation, which is equivalent to the rate of adenosine production, was calculated to be 0.98 nmol/min per g. Adenosine release from the hearts was 40-50 pmol/min per g. (2) Hearts were perfused with hypoxic medium containing [35S]homocysteine (50 microM). Owing to the hypoxia-induced increase in adenosine production, this procedure also results in expansion and labelling of the AdoHcy pool. From the dilution of the specific radioactivity of AdoHcy relative to that of [35S]homocysteine, the rate of AdoHcy synthesis from AdoMet (transmethylation) was calculated to be 1.12 nmol/min per g. It is concluded that in the oxygenated heart the transmethylation pathway is quantitatively an important intracellular source of adenosine, which exceeds the rate of adenosine wash-out by the coronary system by about 15-fold. Most of the adenosine formed by this pathway is re-incorporated into the ATP pool, most likely by adenosine kinase. The transmethylation pathway is essentially O2-independent, and the known hypoxia-induced production of adenosine must be derived from an increase in 5'-AMP hydrolysis.     

Inhibition of phospholipid methylation in isolated rat hepatocytes by analogues of adenosine and S-adenosylhomocysteine

Phospholipid methylation in isolated hepatocytes was inhibited in the presence of 3-deazaadenosine (ID₅₀ = 1.7 μM) 9-β-d-arabinofuranosyladenine (ID₅₀ = 6.0 μM), S-tubercidinylhomocysteine (ID₅₀ = 30 μM), and 5′-deoxy-5′-isobutylthioadenosine (ID₅₀ = 177 μM). A transient inhibitory effect was observed with adenosine, whereas S-adenosyl-l-homocysteine and Sinefungin were essentially without effect. The inhibition of phospholipid methylation by S-tubercidinylhomocysteine and 9-β-d-arabinofuranosyladenine showed a lag-phase, whereas the effect of the other inhibitors was apparent within a few minutes. Cells exposed to 9-β-d-arabinofuranosyladenine or 3-deazaadenosine accumulated large amounts of AdoHcy, and adenosine induced a transient increase in the AdoHcy level. In addition, 3-deazaadenosine served as a precursor for the formation of S-3-deazaadenosylhomocysteine, which accumulated rapidly in cells exposed to this agent. The inhibitory effects of 3-deazaadenosine, 9-β-d-arabinofuranosyladenine and adenosine could be explained by the increase in total nucleosidylhomocysteine induced by these agents. In contrast, only a slight (less than 2-fold) increase in S-adenosyl-l-homocysteine content was observed in hepatocytes treated with 5′-deoxy-5′-isobutylthioadenosine, and this metabolic effect could not explain the inhibition of phospholipid methylation induced by this agent. None of the compounds tested reduced the amount nor the specific radioactivity of S-adenosylmethionine. Biological processes determining the inhibitory effects of adenosine, S-adenosyl-l-homocysteine and their analogues on phospholipid methylation in intact cells are discussed.     

Effect of adenosine metabolites on methyltransferase reactions in isolated rat livers

Adenosine is rapidly metabolized by isolated rat livers. The major products found in the perfusate were inosine and uric acid while hypoxanthine could also be detected. S-Adenosylhomocysteine was also excreted when the liver was perfused with both adenosine and L-homocysteine. A considerable portion of the added adenosine was salvaged via the adenosine kinase reaction. The specific radioactivity of the resultant AMP reached 75–80% of the added [8-¹⁴C]adenosine within 90 min. When the liver was perfused with adenosine alone, hydrolysis of S-adenosyllhomosysteine, via S-adenosylhomocysteine hydrolase, appeared to be blocked resulting in the accumulation of this compound. As the intracellular level of S-adenosylhomocysteine increased, the rates of various methyltransferase reactions were reduced, resulting in elevated levels of intracellular S-adenosylmethionine. When the liver was perfused with normal plasma levels of methionine the S-adenosylmethionine : S-adenosylhomocysteine ratio was 5.3 and the half-life of the methyl groups was 32 min. Upon further addition of adenosien the S-adenosylmethionine : S-adenosylhomocysteine ratio shifted to 1.7 and the half-life of the methyl groups to 103 min. In the presence of adenosine and L-homocysteine such inordinate amounts of S-adenosylhomocysteine accumulated in the cell that methylation reactions were completely inhibited. Although adenine has been found to be a product of the S-adenosylhomocysteine hydrolase only trace quantities of this compound were detectable in the tissue after perfusing the liver with high concentrations of adenosine for 90 min.     

Inhibition of phosphoinositide metabolism in human polymorphonuclear leukocytes by S-adenosylhomocysteine

Transmembrane signaling by chemoattractants in leukocytes appears to require activation of phosphoinositide metabolism with subsequent generation of the second messenger substances, inositol(1,4,5)trisphosphate and diacylglycerol. In addition, previous studies have shown that conditions which lead to an intracellular increase in S-adenosylhomocysteine (AdoHcy), a by-product and competitive inhibitor of S-adenosylmethionine-mediated methylation reactions, inhibit all chemoattractant-mediated functions of leukocytes, suggesting that AdoHcy also interferes with chemoattractant transmembrane signaling. In the present study, we determined whether AdoHcy altered the metabolism of phosphoinositides in human polymorphonuclear leukocytes. Treatment of 32P-labeled polymorphonuclear leukocytes with the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, plus exogenous adenosine and L-homocysteine thiolactone, conditions which cause an increase in AdoHcy, produced as much as a 37% decrease in the amount of [32P]phosphatidylinositol 4-monophosphate associated with the cells. The formation of inositol bisphosphate was inhibited by as much as 45% by erythro-9-(2-hydroxy-3-nonyl)adenine, adenosine, and L-homocysteine thiolactone suggesting decreased availability of phosphatidylinositol 4-monophosphate. In support of this, AdoHcy, in concentrations ranging from 0.01 to 0.1 mM, inhibited the transfer of gamma-32P from gamma-[32P] ATP to phosphatidylinositol (PtdIns). The inhibition of PtdIns kinase was competitive with an apparent Ki for AdoHcy of 43 microM. Increased intracellular AdoHcy reduced chemoattractant-mediated increases in inositol(1,4,5)trisphosphate formation suggesting abrogation of transmembrane signaling. These findings for the first time demonstrate that AdoHcy is a competitive inhibitor of PtdIns kinase and thus a regulator of the phosphoinositide pathway.     

Role of S-adenosylhomocysteine hydrolase in adenosine metabolism in mammalian heart.

S-Adenosylhomocysteine hydrolase of mammalian hearts from different species is exclusively a cytosolic enzyme. The apparent Km for the guinea-pig enzyme was 2.9 microM (synthesis) and 0.39 microM (hydrolysis). Perfusion of isolated guinea-pig hearts for 120 min with L-homocysteine thiolactone (0.23 mM) and adenosine (0.1 mM), in the presence of erythro-9-(2-hydroxynon-3-yl)adenine to inhibit adenosine deaminase, caused tissue contents of S-adenosylhomocysteine to increase from 3.5 to 3600 nmol/g. When endogenous adenosine production was accelerated by perfusion of hearts with hypoxic medium (30% O2), L-homocysteine thiolactone (0.23 mM) increased S-adenosyl-homocysteine 17-fold to 64.3 nmol/g within 15 min. In the presence of 4-nitro-benzylthioinosine (5 microM), an inhibitor of adenosine transport, S-adenosylhomocysteine further increased to 150 nmol/g. L-Homocysteine thiolactone decreased the hypoxia-induced augmentation of adenosine, inosine and hypoxanthine in the tissue and the release of these purines into the coronary system by more than 50%. Our findings indicate that L-homocysteine can profoundly alter adenosine metabolism in the intact heart by conversion of adenosine into S-adenosylhomocysteine. Adenosine formed during hypoxia was most probably generated within the myocardial cell.     

The rate of transmethylation in mouse liver as measured by trapping S-adenosylhomocysteine

Periodate-oxidized adenosine has previously been shown to be a potent inhibitor in vitro of S-adenosylhomocysteine hydrolase (E.C. 3.3.1.1). This paper describes the inhibition of this enzyme in liver following injection of mice with periodate-oxidized adenosine. A maximally effective dose of 100 nmol/g of this compound causes liver S-adenosylhomocysteine to increase from 12 to 600 nmol/g within 30 min. This accumulation of S-adenosylhomocysteine provides an estimate of the rates of transmethylation, as well as adenosine and homocysteine production, as being at least 20 nmol/min/g liver. A doubling of S-adenosylmethionine in the liver of mice treated with periodate-oxidized adenosine suggests that the high levels of S-adenosylhomocysteine inhibit some transmethylation reactions.     

Role of S-adenosylhomocysteine in adenosine-mediated toxicity in cultured mouse T lymphoma cells

S-adenosylhomocysteine (SAH) is known to be a potent inhibitor of S-adenosylmethionine (SAM)-mediated reactions, of which SAH itself is a product. The immediate metabolic fate of SAH involves its hydrolysis to adenosine and L-homocysteine by the enzyme SAH hydrolase, but the reversibility of this reaction and its extremely low K_eq in the hydrolytic direction suggest that under certain conditions of adenosine excess, SAH might accumulate with significant cytotoxic effects. We have used a model system consisting of cultured S49 mouse lymphoma cells together with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), to determine whether SAH is a mediator of adenosine cytotoxicity.Cells rendered resistant to adenosine-induced pyrimidine starvation by the addition of exogenous uridine or by the mutational loss of adenosine kinase are still sensitive to adenosine at concentrations >15 μM. We find that this effect is appreciably enhanced by the addition of L-homocysteine thiolactone to the culture medium. Cytotoxic concentrations of adenosine also cause significant elevations in intracellular levels of SAH, which are increased an additional several fold by 100μM exogenous L-homocysteine thiolactone. A fair correlation exists between a single time point determination of intracellular SAH and the degree of growth inhibition after 72 hr, but complicated time-dependent variations in SAH make it difficult to compare results obtained in the absence and presence of exogenous L-homocysteine thiolactone.In vivo DNA methylation in S49 cells is markedly inhibited by exposure of cells to concentrations of adenosine known to cause uridine-resistant cytotoxicity. This inhibition of methylation has been measured with short-term pulses of radiolabel, and correlates well with intracellular concentrations of SAH at all tested combinations of adenosine and L-homocysteine thiolactone. The results suggest that the uridine-resistant cytotoxic effects of adenosine on ADA-inhibited S49 cells are secondary to the inhibition of SAM-mediated methylation reactions by the adenosine metabolite SAH.     

New Neplanocin Analogues. V. A Potent Adenosylhomocysteine Hydrolase Inhibitor Lacking Antiviral Activity. Synthesis And Antiviral Activity Of 6″-Carboxylic Acid Derivatives Of Neplanocin A 1

Abstract

The 6′-carboxylic acid derivative of neplanocin A 3 was synthesized from NPA, and was converted to the corresponding methyl ester 4 and amides 5 and 6. These were evaluated for their anti-RNA-virus activities. Of the derivatives synthesized, only 5 was active against RNA viruses within the concentration range of 0.14-4.88 μg/mL. Compounds 3 and 5 showed a potent inhibitory effect on S-adenosylhomocysteine (AdoHcy) hydrolase from rabbit erythrocytes. Although a close correlation between the inhibitory effect of adenosine analogues on AdoHcy hydrolase and their antiviral potency has been demonstrated, 3 did not show any anti-RNA-virus activities.

     

Characterization of three rabbit liver lysosomal proteinases with fructose 1,6-bisphosphatase converting enzyme activity

A large number of nucleoside analogs have been found to inactivate S-adenosylhomocysteine (AdoHcy) hydrolase in a time-dependent irreversible manner. There are two classes of these irreversible inhibitors: (A) analogs that inactivate the enzyme in a pseudofirst-order process and are devoid of any side chain at the 5′-OH group; (B) analogs that inactivate the enzyme in a time-dependent but curvilinear process, and generally have a side chain at the 5′ position. Among the more potent irreversible inhibitors are 2-chloroadenosine, 9-β-d-arabinofuranosyladenine (Ara-A), and (±)aristeromycin. Release of adenine base from adenosine or Ara-A in the presence of AdoHcy hydrolase was observed, thus supporting the proposed catalytic mechanism of AdoHcy hydrolase, that entails the transient formation of 3′-ketoadenosine during enzymatic catalysis of either the formation or hydrolysis of AdoHcy. Both Ara-A and adenosine may exert their irreversible inactivation by a suicide mechanism, but nucleosides such as 5′-iodo-5′-deoxyadenosine and 3′-deoxyadenosine are probably strictly irreversible inhibitors per se in view of the catalytic mechanism proposed for AdoHcy hydrolase. Labeling of AdoHcy hydrolase, perhaps covalent in nature, by radioactive Ara-A and adenosine was demonstrated by gel electrophoresis.     

Interaction between adenosine generated endogenously in neocortical tissues,and homocysteine and its thiolactone

[¹⁴C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted ¹⁴C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [¹⁴C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [¹⁴C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [¹⁴C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide ¹⁴C-derivative when homocysteine was 0.1 mM or greater. The total [¹⁴C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.     

Myocardial adenosine cycling rates during normoxia and under conditions of stimulated purine release.

Formation and rephosphorylation of adenosine (adenosine cycling) was studied in isolated rat hearts during normoxia and under conditions of stimulated purine formation. Hearts were infused with an inhibitor of adenosine kinase (5-iodotubercidin, 2 microM). In addition, perfusions were carried out with or without acetate, which is converted into acetyl-CoA, with simultaneous breakdown of ATP to AMP and purines. We found a linear, concentration-dependent, increase in normoxic purine release by acetate (5-20 mM). Differences in total purine release with or without iodotubercidin were taken as a measure of adenosine cycling. In normoxic hearts, iodotubercidin caused a minor increase in purine release (2.7 nmol/min per g wet wt.). Acetate (12.5 mM) increased purine release by 4.9 nmol/min per g, and its combination with inhibitor gave a large increase, by 14.2 nmol/min per g. This indicates a strongly increased adenosine cycling rate during acetate infusion. However, no significant differences in purine release were observed when iodotubercidin was infused during hypoxia, anoxia or ischaemia. The hypothesis that adenosine cycling is near-maximal during normoxia was not confirmed. Increased myocardial adenosine formation appears to be regulated by the availability of AMP and not by inhibition of adenosine kinase. This enzyme mainly functions to salvage adenosine in order to prevent excessive loss of adenine nucleotides.     

Synthesis and antiviral activities of 3-deaza-3-fluoroaristeromycin and its 5′ analogues

The naturally occurring adenine based carbocyclic nucleosides aristeromycin and neplanocin A and their 3-deaza analogues have found a prominent place in the search for diverse antiviral activity agent scaffolds because of their ability to inhibit S-adenosylhomocysteine (AdoHcy) hydrolase. Following the lead of these compounds, their 3-deaza-3-fluoroaristeromycin analogues have been synthesized and their effect on S-adenosylhomocysteine hydrolase and RNA and DNA viruses determined.     

Homocysteine in tissues of the mouse and rat

A method for the determination of L-homocysteine (Hcy) in tissues is described, which involves adsorption of adenosine and S-adenosyl-L-homocysteine (AdoHcy) in the tissue extract to dextran-coated charcoal, while leaving Hcy in solution. Sufficient dilution of the tissue homogenates and the presence of a reducing agent during the adsorption step are required to obtain high recovery of Hcy. Hcy is condensed with radioactive adenosine, and labeled AdoHcy is quantified by high performance liquid chromatography on a 3-micron reversed phase column. The amount of Hcy was determined in several tissues (liver, kidney, brain, heart, lung, and spleen) of mice and rats, and the concentrations of Hcy were in the range 0.5-6 nmol/g, wet weight. Hcy concentration was about 1 microM in mouse plasma. In mice, liver contained the highest amount of Hcy, and kidneys were also rich in Hcy. Similar concentrations were found in rat tissues. S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), the enzyme which is believed to catalyze the only pathway leading to Hcy formation in vertebrates, was nearly completely inactivated in mice injected with the drug combination 9-beta-D-arabinofuranosyladenine plus 2'-deoxycoformycin. This treatment induced a massive accumulation of AdoHcy in all tissues (Helland, S., and Ueland, P. M. (1983) Cancer Res. 43, 1847-1850). The amount of Hcy increased several-fold in kidney, whereas no change was observed in liver, heart, brain, lung, and spleen.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

Boron deprivation decreases liver S-adenosylmethionine and spermidine and increases plasma homocysteine and cysteine in rats

Two experiments were conducted with weanling Sprague–Dawley rats to determine whether changes in S-adenosylmethionine utilization or metabolism contribute to the diverse responses to boron deprivation. In both experiments, four treatment groups of 15 male rats were fed ground corn-casein based diets that contained an average of 0.05 mg (experiment 1) or 0.15 mg (experiment 2) boron/kg. In experiment 2, some ground corn was replaced by sucrose and fructose to increase oxidative stress. The dietary variables were supplemental 0 (boron-deprived) or 3 (boron-adequate) mg boron/kg and different fat sources (can affect the response to boron) of 75 g corn oil/kg or 65 g fish (menhaden) oil/kg plus 10 linoleic acid/kg. When euthanized at age 20 (experiment 1) and 18 (experiment 2) weeks, rats fed the low-boron diet were considered boron-deprived because they had decreased boron concentrations in femur and kidney. Boron deprivation regardless of dietary oil increased plasma cysteine and homocysteine and decreased liver S-adenosylmethionine, S-adenosylhomocysteine, and spermidine. Plasma concentration of 8-iso-prostaglandin F_2α (indicator of oxidative stress) was not affected by boron, but was decreased by feeding fish oil instead of corn oil. Fish oil instead of corn oil decreased S-adenosylmethionine, increased spermidine, and did not affect S-adenosylhomocysteine concentrations in liver. Additionally, fish oil versus corn oil did not affect plasma homocysteine in experiment 1, and slightly increased it in experiment 2. The findings suggest that boron is bioactive through affecting the formation or utilization of S-adenosylmethionine. Dietary fatty acid composition also affects S-adenosylmethionine formation or utilization, but apparently through a mechanism different from that of boron.     

S-adenosylhomocysteine hydrolase from hamster liver: Purification and kinetic properties

3-Deazaadenosine is both an inhibitor of and a substrate for S-adenosylhomocysteine hydrolase. Its administration to rats results in the accumulation of both S-adenosylhomocysteine and 3-deazaadenosylhomocysteine in the liver and other tissues. In hamsters, however, the administration of 3-deazaadenosine results only in the accumulation of 3-deazaadenosylhomocysteine (P. K. Chiang and G. L. Cantoni (1979) Biochem. Pharmacol. 28, 1897). In order to investigate the possible reasons for this difference, S-adenosylhomocysteine hydrolase from hamster liver has been purified to homogeneity and some of its kinetic and physical parameters have been determined. The molecular weight of the native enzyme is 200,000 with a subunit molecular weight of 48,000. The K_m's for adenosine and 3-deazaadenosine are about 1.0 μm, and the V_max's are also similar. The K_m for S-adenosylhomocysteine is 1.0 μm, or more than 10 times smaller than the K_m of the rat liver enzyme. This difference in K_m value may explain the differences in the response of rat and hamster liver to the administration of 3-deazaadenosine. S-Adenosylhomocysteine hydrolase from hamster liver exhibits an interesting kinetic property in that its activity can be affected bimodally by either adenosine or adenosine Anal.ogs. At very low concentrations of these analogs, the activity of S-adenosylhomocysteine hydrolase can be stimulated by 10–30%, and at higher concentrations these same analogs become competitive inhibitors.     

Does uptake of Alexandrium fundyense cysts contribute to the levels of PSP toxin found in the sea scallop, Placopecten magellanicus?

Atlantic sea scallops, Placopecten magellanicus, in most areas of the Bay of Fundy, New Brunswick, Canada, have year-round concentrations of paralytic shellfish posioning (PSP) toxins greater than the regulatory concentration of 80 μg STX eq. 100 g⁻¹ wet weight. Scallops (mean shell height of 10.7 cm, age 3–5 years) were collected by SCUBA and individually tagged near Parker Island, Bay of Fundy. Half were hung 2 m below the low tide water level and the remainder were placed on the bottom (11 m depth at low tide) under the scallops held at 2 m. Scallop, water and sediment samples were collected monthly for determination of concentrations of PSP toxins and Alexandrium fundyense.In October, 1993, mean concentrations of PSP toxins in digestive gland, and mantle were 3205 and 1018 μg STX eq. 100 g⁻¹ wet weight, respectively. Eight months later (June 1994), PSP concentrations in digestive glands from the surface and bottom had declined to 504 and 682 μg STX eq. 100 g⁻¹ wet weight, respectively, whereas those in the mantle had declined to 802 and 681 μg STX eq. 100 g⁻¹ wet weight. During July 1994, A. fundyense concentrations observed at Parker Island and offshore were 320 cells l⁻¹ and 14,200 cells l⁻¹, respectively. Subsequently, toxin concentrations in surface and bottom scallop digestive glands increased to 12,720 and 11,408 μg STX eq. 100 g⁻¹ wet weight, whereas concentrations in mantles increased to 2126 and 1748 μg STX eq. 100 g⁻¹ wet weight, respectively. Concentrations of PSP toxins in these tissues in October 1994 were similar to those measured in October 1993. Concentrations of PSP toxin were less than the regulatory concentration in the gonads and non-detectable in adductor muscles of all scallops sampled.There were no statistically significant differences in profiles for uptake and depuration of PSP toxins in scallops held at the surface compared to those from bottom, suggesting that A. fundyense cysts at the concentrations found in the sediment (45 cysts cm⁻³) did not contribute significantly to the year-round presence of PSP toxins within scallop tissues. The year-round occurrence of PSP toxin is probably due to accumulation during summer blooms followed by a very slow rate of depuration.     

New Δ8(14)-15-Ketosterols with an Oxygenated Side Chain

New analogues of 3β-hydroxy-5α-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3β-hydroxy-24-methyl-22,23-oxido-5α -cholest-8(14)-en-15- ones and (22RS,23ξ,24S)-24-methyl-5α-cholesta-8(14)-ene-3β, 22,23-triol-15-one] were synthesized from (22E,24S)-3β-acetoxy-24-methyl-5α-cholesta-8(14), 22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC₅₀ 0.9±0.2 and 0.7±0.2 μM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC₅₀ 4.0±0.5 μM), (22E,24S)-3β-hydroxy-24-methyl-5α-cholesta-8(14),22- dien-15-one (IC₅₀ 3.1±0.4 μM), and the 3β,22,23-triol synthesized (IC₅₀ 6.0±1.0 μM).__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 312–319.Original Russian Text Copyright © 2005 by Flegentov, Piir, Medvedeva, Tkachev, Timofeev, Misharin.