Nitric oxide signaling in ischemic heart.

OBJECTIVE: Several recent studies have implicated a role of endogenous nitric oxide (NO) in the pathophysiology of myocardial ischemic/reperfusion injury. However, the mechanism by which NO exerts its beneficial/detrimental effects remains unknown. This study examined the intracellular signaling of NO by studying the role of the NO-cGMP signaling pathway on the phospho-diesteratic breakdown and turnover of phosphoinositides during myocardial ischemia and reperfusion. METHODS: Isolated working rat hearts were made ischemic for 30 min followed by 30 min of reperfusion. A separate group of hearts were pre-perfused with 3 mM L-arginine for 10 min prior to ischemia. The release of NO was monitored using an on-line amperometric sensor. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [3H]myoinositol and [14C]arachidonic acid. Hearts were then perfused for 10 min in the presence or absence of L-arginine via the Langendorff mode. Ischemia was induced for 30 min followed by 30 min of reperfusion. Experiments were terminated before L-arginine and after L-arginine treatment, after ischemia, and during reperfusion. Biopsies were processed to determine the isotopic incorporation into various phosphoinositols as well as phosphatidic acid and diacylglycerol. cGMP was assayed by radioimmunoassay and SOD content was determined by enzymatic analysis. RESULTS: The release of NO was diminished following ischemia and reperfusion and was augmented by L-arginine. L-Arginine reduced ischemic/reperfusion injury as evidenced by the enhanced myocardial functional recovery. cGMP, which remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The cGMP level persisted up to 10 min of reperfusion and then dropped slightly. Reperfusion of ischemic myocardium resulted in significant accumulation of radiolabeled inositol phosphate, inositol bisphosphate, and inositol triphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly during reperfusion. Reperfusion of the ischemic heart prelabeled with [14C]-arachidonic acid resulted in modest increases in [14C]diacylglycerol and [14C]phosphatidic acid. Pretreatment of the heart with L-arginine significantly reversed this enhanced phosphodiesteratic breakdown during ischemia and early reperfusion. However, at the end of the reperfusion the inhibitory effect of L-arginine on the phosphodiesterases seems to be reduced. In L-arginine-treated hearts, SOD activity was progressively decreased with the duration of reperfusion time. CONCLUSIONS: The results suggest for the first time that NO plays a significant role in transmembrane signaling in the ischemic myocardium. The signaling seems to be transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. This signaling effect appears to be reduced as reperfusion progresses. These results, when viewed in the light of free radical chemistry of NO, suggest that such on- and off-signaling of NO may be linked to its interaction with the superoxide radical generated during the reperfusion of ischemic myocardium.

The red wine antioxidant resveratrol protects isolated rat hearts from ischemia reperfusion injury.

The consumption of red wine has been reported to impart a greater benefit in the prevention of coronary heart disease than the consumption of other alcoholic beverages. This beneficial effect is increasingly being attributed to certain antioxidants comprising the polyphenol fraction of red wine such as transresveratrol. In the present study, we investigated the potential cardioprotective effects of resveratrol in the face of ischemia reperfusion (I/R) injury. Isolated perfused working rat hearts after stabilization were perfused with Krebs-Henseleit Bicarbonate buffer (KHB) either in the presence or absence of transresveratrol (RVT) at a concentration of 10 microM for 15 min prior to subjecting them to 30 min of global ischemia followed by 2 h of reperfusion. Left ventricular functions were monitored at various timepoints throughout the reperfusion period to assess the extent of postischemic recovery in comparison with baseline values. Coronary perfusate samples were also collected to determine malonaldehyde (MDA) levels. The results demonstrated that RVT exhibited significant myocardial protection. This was evidenced by improved recovery of post-ischemic ventricular function including developed pressure and aortic flow as compared to the control group (KHB). Values for developed pressure in the RVT-treated group were significantly higher than those in the control group throughout the reperfusion period (71.09+/-4.88 mm Hg vs. 58.47+/-3.88 mm Hg, 68.87+/-5.07 mm Hg vs. 49.74+/-2.65 mm Hg and 51.67+/-3.95 mm Hg vs. 30.50+/-4.80 mm Hg at reperfusion timepoints R-15, R-60, and R-120, respectively). From R-30 onwards, aortic flow was markedly higher in the RVT treated group as compared with the control group, the differences being most significant at R-90 (32.45+/-2.19 ml/min vs. 19.83+/-1.62 ml/min) and R-120 (27.15+/-2.27 ml/min vs. 14.10+/-1.69 ml/min). In contrast to the KHB treated group, the RVT-treated group displayed significant reduction in MDA formation especially in the immediate early reperfusion period (63.71+/-8.19 pM/ml vs. 130.86+/-4.76 pM/ml, 63.84+/-15.62 pM/ml vs. 156.99+/-18.93 pM/ml, 71.29+/-2.80 pM/ml vs. 129.5+/-10.30 pM/ml and 56.25+/-5.79 pM/ml vs. 127.99+/-3.50 pM/ml at timepoints R-1, R-3, R-5, and R-7, respectively) indicating a reduction in I/R injury related oxidative stress. Infarct size was markedly reduced in the RVT group when compared with the control group (10.57+/-0.35% vs. 36.27+/-5.28%). In vitro studies revealed RVT to be a potent scavenger of peroxyl radicals suggestive of a probable mechanism involved in the protective ability of RVT. The results of this study indicate that resveratrol possesses cardioprotective effects which may be attributed to its peroxyl radical scavenging activity.

Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts.

While much is known about the beneficial effects of myocardial stress adaptation, relatively less information is available about the adaptive mechanisms. To explore the signaling pathways of stress adaptation, isolated working rat hearts were divided into three groups. Group I was adapted to stress by conventional technique of repeated ischemia and reperfusion consisting of 5 min of ischemia followed by 10 min of reperfusion, repeated four times. Group II was treated with 100 microM of genistein, a tyrosine kinase inhibitor, followed by preconditioning as described for group I. The third group, perfused with buffer only for 60 min, served as control. All hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. The results of our study demonstrated better postischemic myocardial functions in the preconditioned hearts as evidenced by increased aortic flow, coronary flow, developed pressure and lesser amount of tissue injury as evidenced by the decreased creatine kinase release. The preconditioning effects were associated with enhancement of phospholipase D activity in the heart. The preconditioning effect was almost abolished by the genistein treatment which also prevented the enhancement of phospholipase D activities. Additionally, preconditioning of the rat hearts stimulated protein kinase C, MAP kinase, and MAPKAP kinase 2 activities which were inhibited by genistein. The results identifies for the first time tyrosine kinase-phospholipase D as potential signaling pathway for ischemic preconditioning, and implicates the involvement of multiple protein kinases in myocardial adaptation to ischemia.

Comparisons of ESR and HPLC methods for the detection of OH. radicals in ischemic/reperfused hearts. A relationship between the genesis of free radicals and reperfusion arrhythmias.

In this study we compared two methods, electron spin resonance (ESR) spectroscopy and high performance liquid chromatography (HPLC), which are currently used to detect directly hydroxyl radical (OH.) formation in the ischemic and reperfused heart. Isolated buffer-perfused rat hearts were subjected to 30 min of normothermic global ischemia followed by 30 min of reperfusion. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) was used as a spin-trap agent to detect OH. radicals by ESR and HPLC. In additional HPLC studies, salicylic acid was infused into the heart for the detection of OH. radicals. In all studies, the effects of superoxide dismutase (SOD) and catalase (CAT) on the OH. generation were examined. The results of our studies indicate that, irrespective of the method, OH. was always detected when an ischemic heart was reperfused and showed ventricular fibrillation. The OH. concentration increased dramatically between 60 and 90 sec of reperfusion, peaked between 180 and 210 sec, and then progressively decreased. In all cases, both SOD and CAT were able to reduce the formation of OH. radicals, with SOD being relatively more effective. Our results indicate that OH. was produced only in the fibrillating hearts that peaked between 180 and 210 sec (1.64 +/- 0.09 nmol/mL measured by ESR), but not in the non-fibrillating hearts. Although SOD or CAT reduced the OH. formation, they had no effects on the incidence of reperfusion-induced ventricular fibrillation (VF) and ventricular tachycardia (VT). However, when SOD (5 x 10(4) IU/L) was coadministered with CAT (5 x 10(4) IU +/- L), the incidence of reperfusion-induced VF (total) and VT was reduced from their control value of 92 and 100 to 33 (P < 0.05) and 50% (P < 0.05), respectively. The results of this study indicate that the HPLC method, as well as ESR, can be used to detect OH. formation in ischemic/reperfused hearts. Because of the convenience, reproducibility and greater sensitivity, the HPLC technique may be more suitable for OH. detection. Our results further suggest the potential therapeutic value of the combination therapy of SOD and CAT for the reduction of reperfusion-induced VF and VT.

Improvement of ischemia-reperfusion-induced myocardial dysfunction by modulating calcium-overload using a novel, specific calmodulin antagonist, CGS 9343B.

The present paper explores the mechanism of calcium-overloaded cardiac cell exocytosis during reperfusion of ischemic myocardium. A novel specific inhibitor of calmodulin, CGS 9343B, was used to pretreat an ischemic heart in an effort to enhance myocardial preservation. The experimental model employed an isolated in situ pig heart subjected to 120 min of ischemic insult by reversibly occluding the left anterior descending coronary artery, the last 60 min being superimposed with global hypothermic cardioplegic arrest. This ischemic episode was followed by 60 min of revascularization. CGS 9343B enhanced post-ischemic myocardial recovery, as judged by improved regional as well as global myocardial functions, better preservation of high-energy phosphate compounds, and reduced release of creatine kinase. Since this compound blocks calmodulin without inhibiting protein kinase C, the results of this study suggest that calmodulin-dependent kinase, rather than protein kinase C, is primarily involved in expressing calcium-overloaded cell exocytosis, and a specific calmodulin antagonist such as CGS 9343B can be used to salvage an ischemic heart from reperfusion injury.

Enhanced myocardial preservation by nicotinic acid, an antilipolytic compound. Improved cardiac performance after hypothermic cardioplegic arrest.

The effect of nicotinic acid, an antilipolytic drug, on myocardial preservation was studied on the basis of cardiac performance after 2 hours of cardioplegic arrest. Isolated in situ pig hearts were subjected to 120 minutes of hypothermic potassium (35 mEq) crystalloid cardioplegic arrest followed by 60 minutes of reperfusion. The experimental group received nicotinic acid 0.08 mmol/L 15 minutes before cardioplegic arrest, whereas the control group received 15 minutes of unmodified perfusion. There was a marked decline in myocardial creatine phosphate levels during cardioplegic arrest in both groups that returned to the baseline level during reperfusion without a significant intergroup difference, and adenosine triphosphate levels remained stable throughout the experiment in both groups. Myocardial oxygen consumption during reperfusion was significantly higher in hearts treated with nicotinic acid, which was consistent with a significantly greater cardiac contractile force as evaluated by isovolumetric left ventricular pressure measurements. There appeared to be less cardiac membrane damage as measured by creatine kinase release during reperfusion, which was significantly inhibited by treatment with nicotinic acid. The present study supports the conclusion that nicotinic acid improves cardiac performance after hypothermic cardioplegic arrest.

Myocardial salvage by chimyl alcohol: possible role of peroxisomal dysfunction in reperfusion injury.

The results of this study suggest that reperfusion of ischemic myocardium may lead to the peroxisomal disorder both functionally and biochemically. An alkyl glycerol such as chimyl alcohol can protect the ischemic heart from the reperfusion injury probably by enhancing the plasmalogen synthesis.

L-arginine reduces endothelial inflammation and myocardial stunning during ischemia/reperfusion.

BACKGROUND: This study evaluated whether the nitric oxide precursor L-arginine could reduce ischemia/reperfusion injury by preventing leukocyte-endothelial interactions. METHODS: Normothermic regional ischemia was induced in the open-chest working pig heart for 30 minutes followed by 90 minutes of reperfusion. A preischemic 10-minute intravenous infusion of 4 mg.kg-1.min-1 of L-arginine (n = 12) was compared with 12 control pigs. Nitric oxide release was measured from the coronary sinus using an amperometric probe. Left ventricular function, malonaldehyde, creatine kinase, myocardial oxygen extraction, and the soluble adhesion molecules (intracellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1) were measured. RESULTS: Nitric oxide release was significantly reduced from baseline throughout ischemia/reperfusion only in the control group. Systolic and diastolic function, and myocardial oxygen extraction were also significantly decreased during early reperfusion in the control compared with the L-arginine group. Peak creatine kinase release was not significantly different between groups. The incidence of ventricular fibrillation, malonaldehyde release, and soluble intracellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1 were each significantly decreased during reperfusion in the L-arginine group. CONCLUSIONS: L-Arginine reduced lipid peroxidation, plasma levels of soluble adhesion molecules, myocardial stunning, and arrhythmias. These results support an excessive endothelial injury/inflammatory response after regional ischemia/reperfusion that can be ameliorated through augmented nitric oxide.

High-performance liquid chromatographic peak identification of 2,4-dinitrophenylhydrazine derivatives of lipid peroxidation aldehydes by photodiode array detection.

Malonaldehyde (MDA), a product of lipid peroxidation, is a presumptive marker for the development of oxidative stress in tissues and plasmas. In this study we report the photodiode array detection of the 2,4-dinitrophenylhydrazine (DNPH) derivatives of MDA using HPLC. Oxidative stress was produced by injecting (i.p.) bacterial lipopolysaccharide (LPS) into rats at a dose of 100 micrograms/kg, or i.v. into rabbits (1 microgram/kg), or added to freshly drawn human blood (200 ng/ml). Blood was collected at several time points up to 5 h, centrifuged, and equal volumes of 20% TCA were used to precipitate proteins from the plasma. The supernatants were derivatized with DNPH, and the aldehyde-DNPHs were extracted with pentane. After evaporation, aliquots of 10 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column, chromatographed with an acetonitrile-water-acetic acid gradient mobile phase and scanned using Waters 996 photodiode array detector. Peak identification and homogeneity was determined by comparing the experimental peaks and UV scans with those of authentic standards. A significant increase in the DNPH derivative of malonaldehyde (MDA-DNPH), but not of the other aldehyde-DNPH derivatives of formaldehyde (FDA), acetaldehyde (ADA), acetone and propionaldehyde (PDA) was seen over the first hour after LPS administration in anesthetized rats, while in conscious rabbits this trend lasted up to 3 h. The retention times as well as the UV scans of the derivatized aldehydes matched the authentic standards. Thus, photodiode array detection has proved valuable in establishing this HPLC method for estimating oxidative stress. This technique could accurately measure pmol amounts of MDA-DNPH indicating the usefulness of photodiode array detection method for estimating small changes in the oxidative stress.

High-performance liquid chromatographic method for the simultaneous detection of malonaldehyde, acetaldehyde, formaldehyde, acetone and propionaldehyde to monitor the oxidative stress in heart.

Lipid peroxidation (LPO) is the oxidative deterioration of polyunsaturated fatty acids (PUFA) with the production of lipid hydroperoxides, cyclic peroxides, cyclic endoperoxides, and finally fragmentation to ketones and aldehydes (including malonaldehyde, MDA). Estimation of LPO through MDA formation measured by assaying thiobarbituric acid (TBA) reactive products remains the method of choice to study the development of oxidative stress in tissues. However, MDA estimation by TBA reactive products is non-specific and often gives erroneous results. In this report we describe a method using high-performance liquid chromatographic separation to estimate MDA, formaldehyde (FDA), acetaldehyde (ADA), acetone, and propionaldehyde (PDA), the degradation products of oxygen-derived free radicals (ODFR) and PUFA, as presumptive markers for LPO. Oxidative stress was induced in the tissue by perfusing an isolated rat heart with hydroxyl radical generating system (xanthine + xanthine oxidase + FeCl3 + EDTA). The coronary effluents were collected, derivatized with 2,4-dinitrophenylhydrazine (DNPH), and extracted with pentane. Aliquots of 25 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column. The products were eluted isocratically with a mobile phase containing acetonitrile-water-acetic acid (40:60:0.1, v/v/v), measured at three different wavelengths (307, 325 and 356 nm) using a Waters M-490 multichannel UV detector and collected for gas chromatography-mass spectrometry (GC-MS) analysis. The peaks were identified by cochromatography with DNPH derivatives of authentic standards, peak addition, UV pattern of absorption at the three wavelengths, and by GC-MS. The retention items of MDA, FDA, ADA, acetone, and PDA were 5.3, 6.6, 10.3, 16.5, and 20.5 min, respectively. The results of our study indicated progressive increase of all five lipid metabolites as a function of the duration of ODFR perfusion. Hydroxyl radical scavengers, superoxide dismutase plus catalase, completely inhibited the formation of these lipid metabolites, demonstrating that the release of lipid metabolites from the isolated heart was indeed in response to oxidative stress. Since MDA, FDA, ADA, acetone, and PDA are the products of ODFR-PUFA interactions, this method allows proper estimation of LPO which monitors the oxidative stress developed during the reperfusion of ischemic myocardium.

HPTLC analysis of sphingomylein, ceramide and sphingosine in ischemic/reperfused rat heart.

Since the sphingomylein-ceramide-sphingosine pathway, especially ceramide, has been shown to induce programmed cell death (apoptosis), and since apoptosis may be involved with ischemic/reperfused injury in the heart, it became desirable to quantitate the three components in ischemic/reperfused rat heart. One group of rat hearts (n = 6) was isolated and perfused with Krebs-Henseleit buffer using the Langendorff non-recirculating mode. The hearts were perfused for 10 min, made ischemic for 30 min and reperfused for 120 min. Hearts were collected and stored at - 70 degrees C before ischemia, after ischemia and after 30, 60 and 120 min of reperfusion. The hearts were homogenized, and lipids were extracted using the Folch method. The lipids were then chromatographed on Whatman silica gel 60 A high-performance thin-layered chromatography (HPTLC) plates. The plates were developed with iodine, photographed using Photoshop software and quantitated using NIH Imaging software. The results show a 50% decrease of sphingomylein during reperfusion with a corresponding increase in ceramide with sphingosine showing a smaller decrease as compared with the ceramide increase.

Capillary gas chromatography for the assessment of cholesterol oxides in the heart.

Recent studies indicated the presence of cholesterol oxides in biological tissues such as heart under pathophysiological conditions. Currently, no suitable method is available to separate and quantitate these oxides in biological tissues. This study was undertaken to develop a method suitable for the quantification of cholesterol oxides in heart. Since under normal conditions cholesterol oxidation does not occur, we exposed isolated perfused rat heart to oxygen-derived free radicals, which are known to oxidize cholesterol in heart. Free radical treated hearts were homogenized and lipids extracted by conventional techniques. The extracted lipids were saponified, and non-saponified portions were extracted with ether. Silyl derivatives of the extracted products were subjected to capillary gas chromatography. One microlitre of the sample was injected onto an SE 54 Supelco capillary column and run by stepwise temperature programming from 230 to 260 degrees C at a rate of 20 degrees C min-1, and then from 260 to 290 degrees C at 6 degrees C min-1. 5-Androsten-3 beta-ol-17-one was used as internal standard. The separated cholesterol oxides were identified by comparing with authentic standards. This method was suitable to separate 7 alpha-hydroxycholesterol, dihydroxycholesterol, 7 beta-hydroxycholesterol, 20 alpha-hydroxycholesterol, cholesterol-5 alpha,6 alpha-epoxide, and 5-cholestene in heart. Since some of these oxides are cytotoxic to the heart, identification of these oxides should be important to understand the pathophysiology of myocardial disease and for the successful therapy to prevent them.

Fatty acid profiles of plasmalogen choline and ethanolamine glycerophospholipids in pig and rat hearts.

The presence of relatively high concentrations of plasmalogen choline and ethanolamine in the heart of many animal species suggests a role of these ether-linked phospholipids in the pathophysiology of certain myocardial diseases. However, the fatty acid composition of myocardial plasmalogens in many species is not known. This study examined the fatty acid composition of the choline and ethanolamine glycerophospholipids in pig heart and compared the results with those in rat heart. Lipids were extracted from the heart biopsies obtained from pig and rat by standard techniques. Phosphoglycerides were separated by thin-layer chromatography followed by their derivatization into fatty acid methyl esters (FAMEs) and dimethyl acetals (DMAs). FAME and DMA samples were analyzed using gas chromatography-mass spectroscopy. Our results indicate striking differences in the fatty acid composition of both choline and ethanolamine glycerophosphates between rat heart and pig heart. Pig heart ethanolamine glycerophosphates are rich in linoleic acid (18:2) and arachidonic acid (20:4), but low in descosahexenoic (22:6) fatty acids while choline glycerophosphates are poor in both 20:4 and 22:6 fatty acids compared to those in rat hearts.

High-performance liquid chromatographic detection of myocardial prostaglandins and thromboxanes.

Reperfusion of ischemic myocardium is associated with the breakdown of membrane phospholipids and a corresponding increase in arachidonic acid, ultimately resulting in the production of prostaglandins (PGs) and thromboxanes (TXs). However, quantification of these arachidonic acid metabolites has been limited to radioimmunoassay because of their presence in extremely low amounts. In this report, we describe a method suitable to detect sub-picogram levels of 6-keto-PGF1 alpha, PGF1 alpha, PGE2 and TXB2 in myocardial perfusates by high-performance liquid chromatography (HPLC) with a high-gain photomultiplier and a xenon-mercury are lamp. Strong Raleigh scatter of the lamp was eliminated by both interference and long-pass cut-off filters. Improved sample clean-up and HPLC separation were achieved by an HPLC system with an Ultrasphere 3-microns C18 column.

Detection of oxidative stress in heart by estimating the dinitrophenylhydrazine derivative of malonaldehyde.

Accurate estimation of the oxidative stress in heart is necessary because the pathogenesis of many heart diseases are believed to be mediated at least in part from the development of oxidative stress resulting from the generation of oxygen free radicals and reduced antioxidant defense system. The most widely used method for this purpose has been the estimation of malonaldehyde (MDA), a lipid peroxidation product, by the thiobarbituric acid (TBA) reaction method. However, because of the nonspecificity of this method, the results are often erroneous. The present report describes a method using high-performance liquid chromatography (HPLC) to estimate MDA. To develop the oxidative stress, two different models were used: ischaemic-reperfused heart and perfusing the heart with a hydroxyl radical (OH+) generating system. The coronary effluents obtained from the isolated rat heart before ischaemia and during the reperfusion of ischaemic heart, as well as during the perfusion of the heart with the OH+ generating system were collected, derivatized with 2,4-dinitrophenylhydrazine (DNPH) and extracted with pentane. Aliquots of 25 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column. The products were eluted isocratically with a mobile phase containing acetonitrile-water-acetic acid (40:60:0.1, v/v/v), measured at 307 nm using a Waters M-490 multichannel UV detector and collected for gas chromatography-mass spectrometry (GC-MS). The peaks were identified by co-chromatography with DNPH derivatives of authentic standards, peak addition, and by GC-MS. The retention time for MDA-DNPH was 5.3 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a major endonuclease from rat liver nuclei.

A major endonuclease has been purified approximately 800-fold from rat liver nuclei using poly(A) as substrate. The enzyme had a molecular weight of about 50,000, and active fractions were obtained which contained no nucleic acid. Enzymatic activity was optimal between pH 6 and 7 and was totally dependent on the presence of a divalent cation. The reaction was inhibited by high ionic strength, polydextran sulfate, heparin, and sodium pyrophosphate. The purified enzyme readily hydrolyzed poly(A), poly(U), poly(C), and denatured DNA, whereas poly(G) was not degraded, and transfer RNA, ribosomal RNA, and native DNA were hydrolyzed only at relatively slow rates. These data suggest that the enzyme may be specific for single-stranded polynucleotides. The purified enzyme was essentially devoid of exonuclease activity, and the products of exhaustive endonuclease digestion of poly(A) were small oligonucleotides terminated with a 5'-phosphoryl group. Evidence was obtained that this endonuclease is localized in the nucleoplasm. Possible functions for this activity are discussed.

5'-deoxy-5'-thioanalogs of adenosine and inosine 5'-monophosphate: studies with 5'-nucleotidase and alkaline phosphatase.

The interaction of 5'-deoxy-5'-thioadenosine 5'-monophosphate (A(S)MP) and 5'-deoxy-5'-thioinosine 5'-monophosphate (I(S)MP) with snake venom, 5'nucleotidase, and calf intestinal mucosa alkaline phosphatase has been characterized. The substrates, A(S)MP and I(S)MP, are analogs of adenosine 5'-monophosphate and inosine 5'-monophosphate in which sulfur replaces oxygen as the bridge between the 5'-carbon of the ribose and the phosphorous. The P-S bond of both A(S)MP and I(S)MP was hydrolyzed by alkaline phosphatase producing the corresponding thionucleoside as a reaction product. The Km for A(S)MP was 270 microM and the V for alkaline phosphatase was 110 nmol/min/mg (8% of the V for AMP), whereas the corresponding values for I(S)MP were 300 microM and 530 nmol/min/mg protein, respectively. In contrast, 5'-nucleotidase did not catalyze hydrolysis of either A(S)MP or I(S)MP. A(S)MP and I(S)MP were competitive inhibitors of the 5'-nucleotidase hydrolysis of AMP and IMP, respectively, with Ki values of 975 and 13 microM. Decreasing the pH of the reaction from 8.1 to 7.1 lowered the Ki for I(S)MP by 100-fold, to a value of 0.15 microM.