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J Lüthje

Publications and source records attributed to J Lüthje.

18 recordsLinked to original sources
PubMed1989

Identification and partial characterization of an adenosine(5')tetraphospho(5')adenosine hydrolase on intact bovine aortic endothelial cells.

The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.

Acid Anhydride Hydrolases↗
PubMed1989

Origin, metabolism and function of extracellular adenine nucleotides in the blood.

In previous views the role of adenine nucleotides was thought to be confined to the intracellular space of the cell. However, research of the last decades has revealed that nucleotides also occur in the extracellular space. This survey deals with the sources, metabolism and the role in blood of the extracellular adenine mononucleotides ATP, ADP, AMP and the dinucleotides diadenosine tetraphosphate (Ap4A) and diadenosine triphosphate (Ap3A). The latter two are novel compounds, which have recently been discovered in human platelets. The mononucleotides originate from damaged tissues, from red blood cells during haemolysis, from activated platelets, the working muscle and from the nervous system, whereas the dinucleotides are exclusively released from stimulated platelets. Both the adenine mono- and the dinucleotides act as signal molecules on blood cells as well as on cells of the vascular wall, thereby modulating physiological processes such as platelet aggregation, histamine release from mast cells, regulation of vascular tone and white cell functions. In order to limit the signal effects of extracellular nucleotides, blood cells, plasma and the interior of the vessel walls are provided with nucleotide splitting enzymes: ATP, ADP and AMP are mainly degraded by ectoenzymes present on blood cells, endothelial and on smooth muscle cells, whereas dinucleotides are primarily metabolized by plasma enzymes. This review closes with the presentation of the clinical utility of Ap3A and Ap4A as tools for the diagnosis of platelet storage pool defects.

Adenine Nucleotides↗
PubMed1989

Extracellular adenine compounds, red blood cells and haemostasis: facts and hypotheses.

Previously, the role of adenine nucleotides was thought to be confined to the intracellular space of the cell. Research of the last decades has revealed that nucleotides also occur in the extracellular milieu. This survey deals with extracellular adenine compounds in the blood, focussing on their role as chemical mediators in the haemostatic effect of red cells. Erythrocytes may act as pro-aggregatory cells by at least two chemical mechanisms. Firstly, they can enhance platelet aggregation by releasing adenosine diphosphate (ADP), a well known platelet stimulatory substance. ADP is set free when red cells are stressed mechanically, for instance by shear forces generated in the blood stream; ample experimental evidence supporting this view is summarized. Secondly, erythrocytes efficiently take up extracellular adenosine via their nucleoside transporters, thereby removing a potent inhibitor of platelet function. Extracellular adenosine occurs in the blood stream, either directly released from various tissues or as the end product of extracellular adenine nucleotide metabolism, e.g. after degradation of red cell-born ADP or ATP. Finally, a novel mechanism of action of the antithrombotic drug dipyridamole, which has very recently been put forward, is demonstrated. Dipyridamole inhibits platelet function indirectly by blocking the uptake of extracellular adenosine via the nucleoside transporter of red cells; increased adenosine levels in turn are responsible for the antiaggregatory effect of dipyridamole.

Adenine Nucleotides↗
PubMed1988

Levels of 5'-nucleotide phosphodiesterase isoenzymes in normal and pathological sera.

The levels of 5'-nucleotide phosphodiesterase isoenzymes (5'-NPD; EC 3.1.4.1) in sera of 54 healthy donors and 201 inpatients were measured. Isozymes were separated electrophoretically and designated as 5'-NPD-0, -I, -II, -III, -IV and -V in the reverse order of their electrophoretic mobility. In healthy donors all isozymes except 5'-NPD-V were detectable. In pathological sera isozymes 0 to V were elevated in 26.0%, 20.5%, 14.0%, 30.5%, 7.0% and 15.0% of the cases, respectively. Decreased values were found in 6-7%, with the exception of 5'-NPD-IV showing decreased activities in 23.5% of the patients. This average distribution pattern was found in many disorders. However, in diseases of the liver and the pancreas a remarkable accumulation of cases with elevated levels of all isozymes, except 5'-NPD-IV, was observed. All isozymes, except 5'-NPD-IV, showed many significant correlations with other laboratory parameters indicating liver disease. Isozyme IV was not related to these parameters but exhibited a strong correlation with serum albumin. 5'-NPD-II was unproportionally often increased in patients with liver cirrhosis and was the only isozyme with on the average higher levels in women than in men.

Adolescent↗
PubMed1988

Demonstration of a novel ecto-enzyme on human erythrocytes, capable of degrading ADP and of inhibiting ADP-induced platelet aggregation.

The role of ADP as an important inducer of platelet aggregation is generally accepted. Therefore it has been postulated by many authors that the enzymatic removal of extracellular ADP from the circulation is essential to avoid platelet aggregation and thrombus formation. Here we show that erythrocytes essentially contribute to the clearance of ADP. The removal of ADP from suspensions of washed human erythrocytes was due to at least two different activities. One activity, which had already been observed by earlier workers, was identified as adenylate kinase, on the basis of the reaction products and the inhibition by adenosine(5')pentaphospho(5')adenosine (Ap5A). This enzyme was not associated with the cells and was always detectable in cell-free supernatants, indicating that the enzyme had leaked from the cells into the extracellular medium. In contrast, the second activity, which is described here for the first time, was tightly bound to the cells. The activity was not inhibited by Ap5A. The main product of the reaction was AMP, and enzyme activity depended on the presence of divalent cations. The Michaelis constant was about 28 mumol/l. This activity seemed to be an ecto-ADPase. Studies with various inhibitors revealed that degradation of ADP was not due to a non-specific phosphatase. Besides the well known ADPase on the endothelium, the ecto-activity on erythrocytes may play an important part in destroying pro-aggregatory ADP.

Adenosine Diphosphate↗
PubMed1988

Catabolism of Ap4A and Ap3A in whole blood. The dinucleotides are long-lived signal molecules in the blood ending up as intracellular ATP in the erythrocytes.

Adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A) are stored in large amounts in human platelets. After activation of the platelets both dinucleotides are released into the extracellular milieu where they play a role in the modulation of platelet aggregation and also in the regulation of the vasotone. It has recently been shown that the dinucleotides are degraded by enzymes present in the plasma [Lüthje, J. & Ogilvie, A. (1987) Eur. J. Biochem. 169, 385-388]. The further metabolism as well as the role of blood cells has not been established. The dinucleotides were first degraded by plasma phosphodiesterases yielding ATP (ADP) plus AMP as products which were then metabolized to adenosine and inosine. The nucleosides did not accumulate but were very rapidly salvaged by erythrocytes yielding intracellular ATP as the main product. Although lysates of platelets, leucocytes and red blood cells contained large amounts of Ap3A-degrading and Ap4A-degrading activities, these activities were not detectable in suspensions of intact cells suggesting the lack of dinucleotide-hydrolyzing ectoenzymes. Compared to ATP, which is rapidly degraded by ectoenzymes present on blood cells, the half-life of Ap4A was two to three times longer. Since the dinucleotides are secreted together with ADP and ATP from the platelets, we tested the influence of ATP on the rate of degradation of Ap4A. ATP at concentrations present during platelet aggregation strongly inhibited the degradation of Ap4A in whole blood. It is suggested that in vivo the dinucleotides are protected from degradation immediately after their release. They may thus survive for rather long times and may act as signals even at sites far away from the platelet aggregate.

Adenine Nucleotides↗
PubMed1987

Catabolism of Ap4A and Ap3A in human serum. Identification of isoenzymes and their partial characterization.

A hydrolase splitting adenosine (5')triphospho(5')adenosine (Ap3A) and adenosine(5')tetraphospho(5')adenosine (Ap4A) has recently been highly purified from human plasma [Lüthje, J. and Ogilvie, A. (1985) Eur. J. Biochem. 149, 119-127]. This enzyme has been shown to have 5'-nucleotide phosphodiesterase activity (5'-NPD). Three isoenzymes splitting Ap4A and Ap3A were found in human serum by means of native polyacrylamide gel electrophoresis. They exactly comigrated with the 5'-NPD isoenzymes I, III and IV according to published nomenclature, and were designated Ap4Aase isozymes I, III and IV. Their Km values with Ap4A as a substrate were 3 microM, 2 microM and 10 microM, respectively. No Ap4A splitting activity corresponding to 5'-NDP-II was found. Further experiments were designed to prove the identity of Ap4Aases with 5'-NPD isoenzymes. Corresponding isozymes of both activities showed identical behaviour upon delipidation of serum with n-butanol: activities I and III were inactivated, whereas IV remained unaffected. Addition of phosphate stimulated Ap4Aase and 5'-NPD isoenzymes I and III, whereas both activities of isozyme IV were inhibited. Further evidence for the identity was obtained when investigating a series of normal and pathological sera showing decreased as well as increased activities of the single isoenzymes. In all cases Ap4Aase and 5'-NPD isoenzymes showed a linear correlation.

Adenine Nucleotides↗
PubMed1987

Separation of 5'-nucleotide phosphodiesterase isoenzymes of human serum by anion exchange high pressure liquid chromatography.

5'-Nucleotide phosphodiesterase isoenzymes (5'-NPD; E.C. 3.1.4.1) of human serum were separated by anion exchange high pressure liquid chromatography. In sera of healthy donors five activities could be separated, designated as Z, A, B, C and D. Under certain pathological conditions a further isozyme E occurred. With the exception of Z all isoenzymes could be related to the activities first described by Tsou and coworkers in normal sera (Tsou et al. J Histochem Cytochem 1973; 21: 402). These authors identified by means of native gel electrophoresis the isoenzymes 5'-NPD-I, 5'-NPD-II, 5'-NPD-III and 5'-NPD-IV, designated in the reverse order of their electrophoretic mobility. By using this gel technique we characterized the activities A, B, C and D as 5'-NPD-II, -III, -IV, and -I, respectively. Activity E corresponded to an isozyme which did not migrate into the gel (designated as 5'-NPD-O).

Chromatography, High Pressure Liquid↗
PubMed1987

5'-Nucleotide phosphodiesterase isoenzymes in human serum: quantitative measurement and some biochemical properties.

A method based on native PAGE is used for the quantitative measurement of 5'-nucleotide phosphodiesterase isoenzymes (5'-NPD; EC 3.1.4.1) in human serum. In contrast to other techniques this method works with a commercially available substrate. In sera of healthy donors four isozymes could be separated, designated as 5'-NPD-I, 5'-NPD-II, 5'-NPD-III and 5'-NPD-IV in the reverse order of their electrophoretic mobility. When low amounts of serum were applied to the gel, the separation between all four activities was sufficient enough to allow their quantitation. Higher amounts of serum impaired the separation between the isoenzymes I and II. However, even when using high amounts of serum, the quantitation of these activities was possible when taking advantage of some of their biochemical properties which are described herein.

1-Butanol↗
PubMed1987

Unproportionally high concentrations of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) in heavy platelets. Consequences for in vitro studies with human platelets.

Platelets from whole blood were separated into five density subpopulations using a discontinuous Percoll gradient. The content of diadenosine triphosphate (Ap3A), diadenosine tetraphosphate (Ap4A), ADP and ATP were determined in the subfractions. The dinucleotides were directly measured in neutralized, acid-soluble extracts of human platelets with a bioluminescence method not requiring any chromatographic step. When comparing the nucleotide contents of the density subpopulations it became evident that all nucleotides steadily increased with increasing density. Ap3A, Ap4A, ADP and ATP were present in 10-, 7-, 4- and 2-fold higher amounts in the heaviest platelets, respectively, as compared to the subfraction with the lowest density. This finding is practically relevant since the most dense platelet subpopulations may be lost during conventional centrifugation to obtain platelet-rich plasma. Therefore we compared a platelet population obtained from PRP with the platelet population, which had been prepared from whole blood by means of a continuous Percoll gradient. All the four nucleotides investigated were represented in 1.5- to 2-fold higher amounts in the whole blood platelet population. This indicates that PRP does not contain a representative population but lacks part of the large heavy platelets containing the highest amounts of nucleotides.

Adenine Nucleotides↗
PubMed1987

Catalytic activity concentrations of diadenosine tetraphosphate hydrolase in normal and pathological sera.

The levels of diadenosine tetraphosphate hydrolase, a nucleotide pyrophosphatase (EC 3.6.1.9), were measured in human sera with a bioluminescence method. 40 sera of healthy donors and 207 samples obtained from inpatients of a medical clinic were analysed. About two thirds of the patients showed increased hydrolase levels, as compared to the normal donors. Elevated levels were not specific for a certain disease, and they were found in quite different disorders. There was no indication for a dependency of enzyme activity on age or sex of the patients. The comparison of diadenosine tetraphosphate hydrolase with seven other laboratory parameters revealed a highly significant correlation with gamma-glutamyl transferase, suggesting that the liver may be a source of elevated serum activities. There was evidence that the gastrointestinal tract and the pancreas may also be regarded as possible organ sources of the hydrolase.

Acid Anhydride Hydrolases↗
PubMed1985

Highly efficient induction of human platelet aggregation in heparinized platelet-rich plasma by diadenosine triphosphate (Ap3A).

Diadenosine triphosphate (Ap3A), which is a releasable dinucleotide of human platelets, induces platelet aggregation when added to heparinized platelet-rich plasma. The concentration dependence of the dinucleotide is similar to ADP. This finding is fully compatible with our recent report of the low potency of Ap3A in citrated platelet-rich plasma relative to ADP. The aggregatory effect of Ap3A in heparinized versus citrated plasma is reflected in the corresponding rates of Ap3A degradation. In citrated plasma, the hydrolysis of Ap3A is drastically slowed down because the hydrolase needs divalent metal ions. The results strongly support the assumption that the aggregatory effect of Ap3A is mediated by the enzymatic hydrolysis of Ap3A which generates ADP as the ultimate stimulus.

Adenine Nucleotides↗
PubMed1985

Catabolism of Ap3A and Ap4A in human plasma. Purification and characterization of a glycoprotein complex with 5'-nucleotide phosphodiesterase activity.

A hydrolase splitting adenosine(5')triphospho(5')adenosine (Ap3A) to AMP and ADP has recently been detected in human plasma [Lüthje, J. and Ogilvie, A. (1984) Biochem. Biophys. Res. Commun. 118, 704-709]. The enzyme has been purified to apparent homogeneity, as stained in a native polyacrylamide gel. From gel filtration data a Stokes radius of 5.9 nm was calculated, suggesting a molecular mass of about 230 kDa. The presence of the non-ionic detergent Triton X-100 did not change the molecular mass. The hydrolase dissociated to three major protein components (66 kDa; 45 kDa; 16 kDa) during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. Binding of the native enzyme to concanavalin-A--Sepharose and specific inhibition of binding by methyl mannoside indicated that the hydrolase is a glycoprotein. Two of the subunits (66 kDa; 45 kDa) could be affinity-labeled with radioiodinated concanavalin A. Active hydrolase could be prepared in buffers without added metal ions. Treatment with EDTA, however, completely abolished the hydrolytic activity. The enzyme could be reactivated by incubation with Ca2+, Co2+ and, at best, with Zn2+, whereas Mg2+ was ineffective. The affinity of the enzyme for Ap3A was high (Km = 1 microM), with normal Michaelis-Menten kinetics. The homolog dinucleotide Ap4A was also substrate (Km = 0.6 microM) yielding AMP and ATP as products after the asymmetric split. Other dinucleotides, such as NAD, and also mononucleotides (ATP,UTP) were degraded to nucleoside monophosphates indicating a broad specificity of the enzyme. The synthetic compound thymidine 5'-monophosphate p-nitrophenyl ester was substrate with low affinity whereas its 3'-homolog was not hydrolyzed. Optimal activity of the hydrolase was found at pH 8.5.

Adenine Nucleotides↗
PubMed1985

Effects of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) on platelet aggregation in unfractionated human blood.

The effects on platelet aggregation of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A), both of which are stored in and released from platelet granules, have been studied in unfractionated human blood using a microscopic platelet-count ratio method. Ap3A at submicromolar concentrations induces platelet aggregation whereas the homologue dinucleotide Ap4A has disaggregating potency. In the concentration range between 10(-7) to 10(-5) M, Ap3A has been found to be as effective as ADP in triggering aggregate formation. These results confirm and essentially extend our recent findings with platelet-rich plasma that Ap3A is able to trigger platelet aggregation by a slow release of ADP from Ap3A which is catalyzed by a plasma hydrolase. Formation of platelet aggregates was also followed kinetically using a turbidometric method which has been developed for this purpose. In contrast to ADP which very rapidly induces a transient state of aggregation, the effect of Ap3A occurs much more slowly but induces the same maximum of aggregation. The duration of the Ap3A stimulus, however, is longer than that of ADP pointing to a potential physiological function of Ap3A as a "masked" source for ADP.

Adenine Nucleotides↗
PubMed1984

Diadenosine triphosphate (Ap3A) mediates human platelet aggregation by liberation of ADP.

Human platelets store considerable amounts of diadenosine 51,5111-P1, P3-triphosphate, which is released together with the homologue diadenosine tetraphosphate (Ap4A) upon thrombin-induced aggregation (Lüthje,J. & Ogilvie,A. (1983) Biochem. Biophys. Res. Commun. 115, 253-260). We now report that, when added to platelet-rich plasma at 10-20 micron, diadenosine triphosphate gradually induces aggregation. The addition of diadenosine tetraphosphate antagonizes this effect by rapidly disaggregating the platelets. When another physiological but structurally unrelated stimulus, i.e. PAF (Platelet activating factor) is introduced into the system, diadenosine triphosphate drastically enhances and prolongs the aggregatory effect of PAF. Again, Ap4A is antagonistic in this system. The mechanism of Ap3A-stimulation can be explained by the slow and continuous liberation of ADP from Ap3A by the action of a hydrolyzing enzyme which is present in human plasma. Our studies suggest that Ap3A may be physiologically important in providing a relatively long-lived stimulus that can modulate platelet aggregation.

Adenine Nucleotides↗
PubMed1983

The presence of diadenosine 5',5'''-P1,P3-triphosphate (Ap3A) in human platelets.

Diadenosine triphosphate (Ap3A) has been identified and quantified in human platelets using a coupled enzymatic assay specific for Ap3A, after fractionation of acidic extracts with high-performance liquid chromatography. Upon thrombin-induced aggregation, Ap3A is released together with the homologue diadenosine tetraphosphate (Ap4A). Extracts of human platelets do also contain enzymatic activities that degrade diadenosine tetraphosphate as well as diadenosine triphosphate. These enzymes, however, are not released during thrombin-induced aggregation of the platelets.

Acid Anhydride Hydrolases↗
PubMed1981

Influence of dietary nitrogen intake on mammalian branched chain alpha-keto acid dehydrogenase activity.

Male albino rats (100-130 g) were fed diets of varying casein contents for a period of six days. Increasing the casein content from 0.38% to 22% led to an elevation of hepatic branched chain alpha-keto acid dehydrogenase (BCKAD) activity. Km values measured with alpha-ketoisocaproate and alpha-ketoisovalerate as substrates were not influenced by the dietary treatment. Replacing the casein content (22%) of the diet by an equivalent amount of amino acids resulted in a significant decrease in hepatic BCKAD activity. The decrease in Vmax values was not accompanied by changes in Km-values. No enhancement of hepatic enzyme activity was observed when increasing the amino acid content of the diet from 17.5% to 25%. Changes in enzyme activity due to alterations of the dietary nitrogen source were not observed in kidney and brain.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗