Adenosine metabolism and its effect on methylation potential in cultured cells: methodological considerations.

Cell culture models are frequently used to study the role of adenosine in several physiological and pathological processes. In the present study, we have shown that adenosine deaminase activity in medium supplemented with calf serum significantly reduces adenosine concentration in culture medium. In the presence of HepG2 cells, the adenosine concentration in culture medium is decreased much faster, because a large amount of exogenous adenosine is metabolized by cellular enzyme. In order to measure intracellular adenosine, inosine, adenine nucleotides, S-adenosylhomocysteine (AdoHcy) and Sadenosylmethionine (AdoMet) contents, two methods for cell harvesting were compared. First, cells were removed with trypsin/EDTA, second, cells were lysed in cell culture dishes immediately after removing culture medium. Our results show that exact determination of adenosine metabolites requires immediate inactivation of metabolism by cell lysis in culture dishes. Application of adenosine (1mM) resulted in a time-dependent increase in intracellular adenosine, inosine, AMP, ATP, AdoHcy and AdoMet concentration. Since AdoHcy levels increased to a larger extent than AdoMet, the methylation potential, expressed as the ratio of AdoMet/AdoHcy, was reduced from 51.8 (control) to 2.9 (adenosine 1 mM, 2 hrs), suggesting that AdoMet-dependent methylation reactions might be impaired. In conclusion our data demonstrate that extracellular adenosine concentration and intracellular metabolite concentration strongly depend on the methods used to culture and harvest the cells.

Effect of acute hyperhomocysteinemia on methylation potential of erythrocytes and on DNA methylation of lymphocytes in healthy male volunteers.

Homocysteine is a precursor of S-adenosylmethionine (AdoMet) and a metabolite of S-adenosylhomocysteine (AdoHcy). The ratio of AdoMet to AdoHcy, defined as the methylation potential (MP), indicates the flow of methyl groups within the cells. Chronic elevations of total homocysteine (tHcy) in plasma correlate with increased AdoHcy concentrations, decreased MP, and impaired DNA methylation. However, the influence of acute hyperhomocysteinemia on MP is unknown. We induced acute hyperhomocysteinemia in 14 healthy volunteers by oral administration of l-homocysteine (65.1 micromol/kg body wt) in an open, randomized, placebo-controlled two-period crossover study. The kinetics of tHcy in blood and urine, MP in blood, and global DNA methylation in lymphocytes were studied systematically during 48 h. Plasma tHcy concentrations reached a peak at 34 +/- 11 min after an oral load with l-homocysteine and decreased with a half-life of 257 +/- 41 min (means +/- SD). Only 2.3% of the homocysteine dose were recovered in urine. AdoHcy concentrations and MP in whole blood and erythrocytes were not affected by the oral homocysteine load. Furthermore, global DNA methylation in lymphocytes did not change under these conditions. We found no difference between the genotypes of 5,10-methylenetetrahydrofolate reductase in response to the homocysteine load. However, AdoMet content in erythrocytes was significantly higher in the C677T carriers (CT; n = 7) compared with the CC genotype (n = 7). Although chronic elevation of tHcy has been shown to affect MP and DNA methylation, acute elevation of plasma tHcy above 20 micromol/l for 8 h is not sufficient to change MP and to induce DNA hypomethylation in lymphocytes.

Simple and sensitive binding assay for measurement of adenosine using reduced S-adenosylhomocysteine hydrolase.

BACKGROUND: Adenosine has been suggested to play an important role in the regulation of renal function. We developed a simple and sensitive binding assay for the detection of adenosine based on the displacement of [(3)H]adenosine from S-adenosylhomocysteine (SAH) hydrolase in its reduced form. METHODS: SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive. This reduced enzyme retains its ability to bind adenosine with high affinity. To determine adenosine in urine or tissues, samples must be deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchloric acid). RESULTS: The reduced SAH hydrolase bound adenosine with a dissociation constant of 33.0 +/- 2 nmol/L. Displacement of adenosine binding by the adenine 5'-nucleotides, adenine and hypoxanthine, required >1000-fold higher concentrations than adenosine itself. The intra- and interassay imprecision (CV) was <3.9% and 7.8%, respectively, and the values obtained showed acceptable correlation with those by HPLC. CONCLUSIONS: The highly sensitive adenosine-binding protein assay is a simple test that allows detection of adenosine in samples with small volumes without purification, and is in this respect superior to HPLC.

Localization of S-adenosylhomocysteine hydrolase in the rat kidney.

S-adenosylhomocysteine (SAH) hydrolase is a cytosolic enzyme present in the kidney. Enzyme activities of SAH hydrolase were measured in the kidney in isolated glomeruli and tubules. SAH hydrolase activity was 0.62 +/- 0.02 mU/mg in the kidney, 0.32 +/- 0.03 mU/mg in the glomeruli, and 0.50 +/- 0.02 mU/mg in isolated tubules. Using immunohistochemical methods, we describe the localization of the enzyme SAH hydrolase in rat kidney with a highly specific antibody raised in rabbits against purified SAH hydrolase from bovine kidney. This antibody crossreacts to almost the same extent with the SAH hydrolase from different species such as rat, pig, and human. Using light microscopy, SAH hydrolase was visualized by the biotin-streptavidin-alkaline phosphatase immunohistochemical procedure. SAH hydrolase immunostaining was observed in glomeruli and in the epithelium of the proximal and distal tubules. The collecting ducts of the cortex and medulla were homogeneously stained. By using double immunofluorescence staining and two-channel immunofluorescence confocal laser scanning microscopy, we differentiated the glomerular cells (endothelium, mesangium, podocytes) and found intensive staining of podocytes. Our results show that the enzyme SAH hydrolase is found ubiquitously in the rat kidney. The prominent staining of SAH hydrolase in the podocytes may reflect high rates of transmethylation. (J Histochem Cytochem 48:211-218, 2000)

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 microM 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 microM range and are negatively correlated with urine flow-rate.

ATP-Sensitive K+ channel modulator binding to sulfonylurea receptors SUR2A and SUR2B: opposite effects of MgADP.

KATP channels are heteromeric complexes of inwardly rectifying K+ channel subunits and sulfonylurea receptors (SURs). SUR2A and SUR2B, which differ within the carboxyl terminal exon 38, are characteristic for the cardiac and smooth muscle type channels, respectively. Here we compare binding of the tritiated KATP channel opener, [3H]P1075, to membranes from human embryonic kidney (HEK) cells transfected with murine SUR2A and 2B at 37 degrees C. Binding to both SURs required addition of Mg2+ and ATP in the low micromolar range. In the presence of MgATP, micromolar concentrations of MgADP, formed by the ATPase activity of the membrane preparation, increased binding to SUR2A but inhibited binding to SUR2B. Decreasing temperatures strongly reduced [3H]P1075 binding to SUR2A, whereas binding to SUR2B was increased in a bell-shaped manner. Kinetic experiments revealed a faster dissociation of the [3H]P1075-SUR2A complex, whereas the association rate constants for [3H]P1075 binding to SUR2A and 2B were similar. Openers inhibited [3H]P1075 binding to SUR2A with potencies approximately 4 times lower than to SUR2B; in contrast, glibenclamide inhibited [3H]P1075 binding to SUR2A approximately 8 times more potently than to SUR2B. The data suggest that SUR2A and 2B represent the opener receptors of cardiac and vascular smooth muscle KATP channels, respectively, and show that MgADP is an important modulator of opener binding to SUR. The different carboxyl termini of SUR2A and 2B lead to differences in the MgADP dependence and the thermodynamics of [3H]P1075 binding, as well as in the affinities for openers and glibenclamide, underlining the importance of this part of the molecule for KATP channel modulator binding.

Effects of ions on adenosine binding and enzyme activity of purified S-adenosylhomocysteine hydrolase from bovine kidney.

The present investigation was undertaken to determine the effect of various ions on the characteristics of S-adenosylhomocysteine (SAH) hydrolase from bovine kidney. The binding sites of [3H]-adenosine to purified SAH hydrolase were not influenced by phosphate, magnesium, potassium, sodium, chloride or calcium ions at physiological cytosolic concentrations. To test whether NAD+ in the SAH hydrolase is essential for adenosine binding, we prepared the apoenzyme by removing NAD+ with ammonium sulfate. The resulting apoenzyme did not exhibit any [3H]-adenosine binding. Since the apoenzyme was enzymatically inactive, it is suggested that adenosine binds to the active site and not to an allosteric site of the intact enzyme. The kinetics of the hydrolysis and the synthesis of SAH catalyzed by the enzyme SAH hydrolase were measured in the presence and absence of phosphate and magnesium. Phosphate increased the Vmax for both synthesis and hydrolysis. However, only the affinity of adenosine for SAH synthesis was significantly enhanced from 10.1+/-1.3 microM to 5.4+/-0.5 microM by phosphate. This effect was already maximal at a phosphate concentration of 1 mM. All other tested ions were without effect on the enzyme activity. Our results show that phosphate at physiological concentrations shifts the thermodynamic equilibrium of SAH hydrolase in the direction of SAH synthesis. These findings imply that SAH-sensitive transmethylation reactions are inhibited during renal hypoxia when intracellular levels of phosphate, adenosine, and SAH are elevated.

S-adenosylhomocysteine-hydrolase from bovine kidney: enzymatic and binding properties.

In the present study S-adenosylhomocysteine (SAH) hydrolase from the bovine kidney has been purified to apparent homogeneity by standard chromatographic procedures. The purified enzyme was free from adenosine deaminase activity and showed a one-banded pattern in SDS-PAGE with a monomer molecular mass of 47,500. The molecular mass of the native enzyme estimated by gel filtration was about 190,000. The pI was 5.5. For hydrolysis of SAH we found a Km of 5.0 +/- 1.2 microM and a V of 0.25 mumol/min/mg. In the direction of synthesis the Km for adenosine was 5.6 microM and V 0.53 mumol/min/mg. The enzyme activity was inhibited in the presence of adenosine with a Ki = 3 microM. In a second set of experiments we determined the binding characteristics of [3H]-adenosine to purified enzyme. The enzyme bound [3H]-adenosine with three apparent affinities: Kd1 = 6.8 +/- 0.7 nM and Bmax1 = 0.24 +/- 0.04 nmol/mg protein; Kd2 = 387 +/- 41 nM and Bmax2 = 1.4 nmol/mg protein, and Kd3 = 7.05 +/- 0.9 microM and Bmax3 = 9 nmol/mg protein. Binding of 25 nM [3H]-adenosine obeyed a monophasic reaction with a k+1 value of 0.025 min/nM. Dissociation of [3H]-adenosine-SAH hydrolase complex was markedly temperature dependent. After a 240-min incubation at 0 degrees C only 5-10% and at 20 degrees C 75% were displaceable. A fraction of 25% bound [3H]-adenosine was not displaceable by unlabeled adenosine. Our data show that the renal SAH hydrolase exhibits similar enzyme kinetics as the well-characterized SAH hydrolase from liver. The amount of SAH hydrolase present in renal tissues (1.4 nmol/g wet weight) could account almost entirely for the binding of renal tissue adenosine. Finally, we report for the first time a high affinity binding site of SAH hydrolase for adenosine, which remains unexplained at present.

Subtyping of coagulation factor XIIIA.

An extended polymorphism of the coagulation factor XIIIA can routinely be detected in human plasma samples and white cell lysates by isoelectric focusing in polyacrylamide gels containing 3 M urea in the pH range 5-8. Analyses of 184 families with 513 children confirmed the formal model proposed by Suzuki et al. [Am J Hum Genet 1988;43:170-174]. Four common alleles, F XIIIA*1A, 1B, 2A, 2B, at an autosomal locus control the expression of ten phenotypes. On the basis of the population sample from southwest Germany the frequencies of the common alleles F XIIIA*1A, 1B, 2A, 2B were calculated as 0.175, 0.609, 0.011, and 0.205, respectively.

[Detection of phosphoglucomutase in the cornea and corneal cell cultures using isoelectric focusing and enzymatic stain methods]. / Nachweis der Phosphoglukomutase in der Kornea und in Korneazellkulturen mit Hilfe der isoelektrischen Fokussierung und enzymatischer Färbemethoden.

PGM-1 and its isoenzymes had been studied in the human cornea, in cornea cell cultures, in erythrocytes and lymphocytes. Qualitative and quantitative comparisons between cornea of healthy persons and cornea cell cultures of patients with cornea dystrophies showed differences between certain phenotypes and their signals of activity.

Linkage analyses of human peptidase C (PEPC), human factor H (HF), and coagulation factor XIIIB (F13B).

Linkage data on human peptidase C (PEPC), human factor H (HF), and coagulation factor XIIIB (F13B) are presented. The results confirm linkage between HF and F13B (lod = 5.32 at theta = 0.10 in males), and give strong evidence for linkage between PEPC and HF (lod = 5.14 at theta = 0.10 in males) and between PEPC and F13B (lod = 3.55 at theta = 0.10 in males). The claim that PEPA is linked with HF must be withdrawn.

Human factor H (beta 1H-globulin): linkage analysis.

Linkage data on human factor H (HF) and 22 other human genetic markers are presented. Close linkage at theta less than 0.10 can be ruled out for a series of marker systems (Rh, PGM1, ACP1, Jk, Tf, Gc, MNSs, ME2, HLA, GLO1, ORM, Gt, PI, Hp, GPT). Strong evidence for linkage was obtained for peptidase A (PEPA) with lods greater than 3.0 at theta = 0.10 in males and at theta = 0.20 for the sexes combined. From this result the HF locus can be provisionally assigned to chromosome 18.