The discovery of a rapidly metabolized polymeric tetraphosphate derivative of adenosine in perfused rat heart.

The predicted presence in perfused rat hearts of a rapidly metabolized but hitherto unrecognized form of adenosine phosphate has been confirmed by specific radioactive labelling. The properties of the purified compound suggest that it is a heteropolymer of a small organic acid, phosphate and purine nucleoside in the proportions 1:4:1.     

Diguanosinetetraphosphatase from rat liver: Acitivity on diadenosine tetraphosphate and inhibition by adenosine tetraphosphate.

The hydrolysis of diadenosine tetraphosphate, a compound previously described by others to occur in liver at concentrations of around 0.1 mu M, is carried out by a specific enzyme. This enzyme has been partially purified from rat liver extracts, and the following properties have been found. The Km value for diadenosine tetraphosphate is 2 mu M; the products of hydrolysis are ATP and AMP; the Km value for diguanosine tetraphosphate is 2 mu M; none of the following substances were substrates of the enzyme: diadenosine triphosphate, diguanosine di and triphosphates, adenosine tetraphosphate, ATP, ADP, NAD+, NADP+ and bis-p-nitrophenylphosphate. Cyclic AMP was not an inhibitor of the reaction. The enzyme requires Mg2+ ions, is maximally active at a pH value of approximately 8, and has a molecular weight of 22000 as estimated by filtration on Sephadex G-100. The activation energy of the reaction was of 10250 cal times mol-1 (42886 J times mol-1). Particularly striking is the inhibition by adenosine tetraphosphate (Ki equals 48 nM) and guanosine tetraphosphate (Ki equals 14 nM). Other nucleotides tested were also competitive inhibitors with Ki values in the 10--100 mu M range.     

The oligomeric structure of high molecular weight adiponectin

There is great interest in the structure of adiponectin as its oligomeric state may specify its biological activities. It occurs as a trimer, a hexamer and a high molecular weight complex. Epidemiological data indicate that the high molecular weight form is significant with low serum levels in type 2 diabetics but to date, has not been well-defined. To resolve this issue, characterization of this oligomer from bovine serum and 3T3-L1 adipocytes by sedimentation equilibrium centrifugation and gel electrophoresis respectively, was carried out, revealing that it is octadecameric. Further studies by dynamic light scattering and electron microscopy established that bovine and possibly mouse high molecular weight adiponectin is C1q-like in structure.     

Protection of the heart from ischemia by a new oligomeric derivative of prostaglandin E1

A lipophilic oligomeric ester was synthesized from prostaglandin E1. The compound was found to protect a Langendorff perfused rat heart from ischemic insult. After 15 minutes cessation of perfusion, the flow was restarted, and the recovery of contraction was measured. In control experiments, the recovery was 16.1 +/- 7.3%. In the case of pre-ischemic addition of the compound (10 micrograms/ml at 10 minutes before ischemic insult), the recovery was 31 +/- 13.2% (n = 7; the difference was not significant). In post-ischemic addition (10 micrograms/ml), the recovery was 75.9 +/- 9.0% (n = 7; p less than 0.01). The compound was also effective in protecting rat heart myocytes against a 60 minutes anoxia/15 minutes reoxygenation injury as judged by the loss of "rod-shaped" intact myocytes. At a 10 micrograms/ml concentration, the compound protected against the loss of rod-shaped myocytes by 30% in pre-anoxia addition and 35% in post-anoxia addition. The levels of significance in these experiments were p less than 0.001. Possible mechanisms of action of this compound are discussed.     

Properties of rat heart adenosine kinase.

Adenosine kinase was purified 870-fold from rat heart by a combination of gel filtration and affinity chromatography. The preparation was free of purine-metabolizing enzymes that could interfere in the assay of the kinase. A study of the properties of the purified enzyme showed that it is activated by Na+ and K+, it possesses a broad pH optimum between 6 and 8, MgATP is the nucleotide substrate, free Mg2+ is an inhibitor with respect to both MgATP and adenosine, and the enzyme is subject to substrate inhibition by adenosine. The severity of this inhibition increases as the concentration of free Mg2+ increase. The Km for MgATP was calculated to be 0.8 mM and that for adenosine, at likely physiological concentrations of MgATP and free MgCl2, was about 0.2 microM. In vivo the enzyme is likely to be saturated with both MgATP and adenosine. Indeed, the adenosine concentration in rat heart in vivo is probably sufficient to cause substrate inhibition, and this would be increased by an increase in free Mg2+ concentration. Changes in the concentrations of adenosine and free Mg2+ may play a role in modifying the activity of the enzyme in vivo.     

Crystallization and oligomeric structure of rat liver arginase.

Rat liver arginase, a manganese-metalloenzyme, has been crystallized from polyethylene glycol 8000 in N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer at pH 8.5. Crystals form as either cubes or pyramids and belong to space group P3(1) (or P3(2)) with hexagonal unit cell dimensions a = b = 88.9 A, c = 114.8 A, or a = b = 88.5 A, c = 104.5 A; the variation along the c axis does not correlate with the external crystal morphology of cube or pyramid-shaped. X-ray diffraction data are measured to a limiting resolution of 2.4 A. Given the volume constraints of the unit cell it is likely that rat liver arginase is a trimer, with three 35,000 Da monomers in the asymmetric unit. This resolves a persistent ambiguity regarding the oligomeric structure of this enzyme.     

Release of AMP and adenosine from rat heart mitochondria

Release of AMP and adenosine from rat heart mitochondria was studied. The rate of appearance of extramitochondrial adenosine was independent of the extramitochondrial phosphate concentration between 5 and 20 mM. In the absence of exogenous, respiratory substrates or in the presence of glutamate/malate plus rotenone, the rate of appearance of adenosine was relatively low when phosphate was not added. The appearance of extramitochondrial AMP + adenosine was found to be directly proportional to the extra-mitochondrial phosphate concentration. Zn2+ (10 mM) decreased the rate of adenosine appearance by 90% and increased the rate of AMP appearance 6-fold. The mitochondrial preparations dephosphorylated exogenous AMP; this activity was inhibited by 10 mM Zn2+. We conclude that the adenosine appearing in the extramitochondrial space was not due to a direct release from the matrix, but instead was due to adenine nucleotide release with subsequent conversion to adenosine in the extramitochondrial space.     

ATP as a source of interstitial adenosine in the rat heart.

The contribution of neuronal ATP to interstitial adenosine levels was investigated in isolated perfused rat hearts. Ventricular surface transudates, representing interstitial fluid, were analyzed for norepinephrine, ATP, and adenosine. Exocytotic release of norepinephrine was induced by electrical stimulation of cardiac efferents emanating from the stellate ganglion. Ganglion stimulation increased contractility, interstitial norepinephrine, ATP, and adenosine. Interstitial adenosine was 11- to 27-fold higher than interstitial ATP, suggesting that the released ATP is unlikely the only source of adenosine. In the presence of AOPCP (alpha,beta-methyleneadenosine 5'-diphosphate), an ecto-5'-nucleotidase inhibitor, the ganglion-stimulated increase in interstitial ATP and adenosine reached levels similar to those in the absence of AOPCP, also suggesting that adenosine does not derive from extracellular ATP. The perfusate Ca2+ was raised from 1 to 4 mM to determine the importance of the enhanced contractile function on the levels of norepinephrine, ATP, and adenosine. The results were increases in contractility and interstitial norepinephrine, ATP, and adenosine, which were not suppressed with atenolol, indicating a norepinephrine-independent release of ATP and adenosine. Reserpine treatment and administration of guanethidine depleted the catecholamine stores and diminished the catecholamine release, respectively. However, neither agent altered Ca2+-induced increases in ATP and adenosine. It is concluded that the amount of neuronal-derived ATP is low and most likely does not contribute significantly to interstitial levels of adenosine. Furthermore, elevations in interstitial norepinephrine, ATP, and adenosine are associated with neuronal-independent increases in contractile function.     

Electron microscopy of native cartilage oligomeric matrix protein purified from the Swarm rat chondrosarcoma reveals a five-armed structure.

Cartilage oligomeric matrix protein was isolated in the native state from the Swarm rat chondrosarcoma. A crucial step was its selective extraction with EDTA-containing buffer. The purified protein was subjected to electron microscopy using rotary shadowing and negative stain. The images allowed the construction of a structural model. The bouquet-like protein consists of five 28-nm-long arms containing a peripheral globular domain, a flexible strand, and a central assembly domain, where the five arms meet in a cylindrical structure.     

Studies of adenosine incorporation in Langendorff rat heart and rat heart mitochondria

The acid-insoluble product isolated from well-oxygenated Langendorff rat heart after perfusion with [¹⁴C]adenosine was purified by phenol extraction and subjected to specific phosphorolysis by pure polynucleotide phosphorylase. TLC analysis of the reaction mixture showed that ADP was the only radioactive product, proving that the original substance was a polyribonucleotide. Studies of the time course of labelling and of the distribution of the acid-insoluble product between the mitochondrial and nuclear fractions showed that both are labelled even after 1 min at 25 °C, but at short times and low temperature more radioactivity is found in the mitochondria. The kinetics of adenosine incorporation resemble those expected for the labelling of hnRNA and mRNA. Isolated, respiring mitochondria incorporate adenosine and adenine nucleotides into acid insoluble form by a process dependent on oxidative phosphorylation and the adenine nucleotide translocase that is specific for adenine derivatives. The results are discussed in terms of the hypothesis that the polyribonucleotide might be a storage form of adenine nucleotides: it is concluded that the bulk of the labelled product is unlikely to play a major role in energy metabolism.     

Long chain fatty acid inhibition of sodium plus potassium-activated adenosine triphosphatase from rat heart.

Long chain fatty acids were found to inhibit (Na+ + K+)-ATPase prepared from rat heart. Unsaturated and polyunsaturated fatty acids were more inhibitory than saturated fatty acids with myristic acid being the most inhibitory saturated fatty acid tested and linoleic the most inhibitory unsaturated fatty acid. As an example of fatty acid modification of the enzyme, inhibition of (Na+ + K+)-ATPase by oleate was examined. When compared to ouabain, inhibition of (Na+ + K+)-ATPase by oleate was found to be similar in that both were dependent on K+ concentration, but, in contrast to the almost instantaneous inhibition by ouabain, oleate inhibition was a slow process requiring over 20 min incubation at 37 degrees to produce maximum inhibition. Inhibition of rat heart (Na+ + K+)-ATPase by oleate was found to be readily reversible by washout. In the presence of albumin an oleate/albumin molar ratio greater than 7.5 was required for inhibition to occur. The activity of rat heart (Na+ + K+)-ATPase had a temperature optimum above 40 degrees and a discontinuous Arrhenius' plot with a transition temperature of 25 degrees. In the presence of oleate, however, the enzyme's optimum temperature decreased to below 40 degrees, the activation energy of the reaction at temperatures below 25 degrees was lowered from 24.7 kcal/mol to 12.6 kcal/mol and the enzyme had a linear Arrhenius' plot. The possibility of in vivo inhibition of cardiac (Na+ + K+)-ATPase under conditions of elevated fatty acids is discussed.     

Structure of a rapidly formed intermediate in ribonuclease T1 folding.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.     

Altered expression of adenosine receptors in heart of diabetic rat.

Diabetes results in functional, biochemical, and morphological abnormalities in the heart. Some of these changes may be attributed to altered adenosine action. This study aimed to examine the expression level of adenosine receptors (AR) in heart of streptozotocin-induced diabetic rat. Performed analyses revealed detectable levels of A1-AR, A2a-AR, A2b-AR, A3-AR mRNA and protein in whole heart and isolated cardiac myocytes. An increase in A1-AR protein content with no changes in mRNA level was observed in isolated cardiac myocytes. Diabetes resulted in an increase of A3-AR mRNA and protein levels in heart and in cardiac myocytes. The level of A2a-AR mRNA was increased in whole diabetic heart, but it decreased in cardiac myocytes with no detectable changes in protein content. We did not observe any changes in expression level of A2b-AR in diabetic heart and isolated cardiac myocytes. Administration of insulin to diabetic rat for four days resulted in returning of the ARs mRNA and protein to the levels observed in heart of normal rat. These changes in ARs genes expression, and receptors protein content correspond to some abnormalities characteristic of the diabetic heart, suggesting involvement in pathogenesis of diabetic cardiomyopathy.     

The characterization and molecular structure of hepatoproliferin: a liver regeneration factor from rat hepatocytes

Hepatoproliferin (HPF) was purified from regenerating rat livers as an oligomeric entity (big-HPF) from which the monomeric form (small-HPF) could be obtained using disaggregating conditions. By using a solid-phase ion-exchange method, small-HPF was forced to dissociate into two charged ionic species, namely norepinephrine (NE) and a sulfonated disaccharide with a molecular structure consisting of D-glucuronic acid bound to glucosamine 2,6-disulfate by a beta-glycosidic linkage having a beta, 1 --> 4 configuration. Monomeric HPF stemmed from the formation of three electrostatic bonds between the protonated amine groups of three norepinephrines, of which two bind to the deprotonated sulfonic groups of glucosamine 2,6-disulfate and one to the deprotonated carboxylic group of glucuronic acid, to constitute a tightly associated complex with a molecular mass of 1046 Da. This represents one of the two purified isoforms of small-HPF. The other isoform, which has a lower molecular mass of 877 Da, lack one NE, leaving the weaker carboxylic group of glucuronic acid unoccupied, to constitute a more acidic form of HPF.     

Further purification of adenosine kinase from rat heart using affinity and ion-exchange chromatography

Adenosine kinase (EC 2.7.1.20) in a cytoplasmic fraction of rat heart was subjected to 5′-AMP-Sepharose 4B chromatography. The enzyme showed affinity for the column in contrast to adenosine deaminase, and was eluted with adenosine plus MgATP. Fractions containing adenosine kinase were put on a column of DEAE-Sephacel and eluted with a gradient. The enzyme was purified up to 3000-fold (yield 10%). The specific activity exceeded 8000 units per gram of protein and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed only one band. We conclude that the method presented is a simple, quick, and elegant way of purifying myocardial adenosine kinase to virtual homogeneity.     

Specific adenosine binding proteins from rat liver.

Specific adenosine-binding proteins from homogenates of rat liver have been fractionated on a DEAE-cellulose column. Three major peaks have been identified with respect to histone phosphokinase and cAMP and adenosine-binding activities. Peak I contains only histone phosphokinase activity not stimulated by cAMP. Peak II contains histone phosphokinase slightly stimulated by cAMP. Both cAMP- and adenosine-binding activities are found in this fraction. The major adenosine-binding protein is associated with Peak III. Histone phosphokinase in Peak III which also binds cAMP is stimulated 2-fold by 2.5 muM cAMP whereas adenosine at 2.5 X 10(-4)M inhibits these enzymes equally well in each of three peaks. The specificity of adenosine binding is discussed.