Acid Phosphodiesterase Research Articles

Crosstalk Between SMPDL3b and NADPH Oxidases Mediates Radiation-Induced Damage of Renal Podocytes.

Patients undergoing radiotherapy (RT) for various tumors localized in the abdomen or pelvis often suffer from radiation nephrotoxicity as collateral damage. Renal podocytes are vulnerable targets for ionizing radiation and contribute to radiation-induced nephropathies. Our prior work previously highlighted the importance of the lipid-modifying enzyme sphingomyelinase acid phosphodiesterase like 3b (SMPDL3b) in modulating the radiation response in podocytes and glomerular endothelial cells. Hereby, we investigated the interplay between SMPDL3b and oxidative stress in mediating radiation injury in podocytes. We demonstrated that the overexpression of SMPDL3b in cultured podocytes (OE) reduced superoxide anion generation and NADPH oxidase activity compared to wild-type cells (WT) post-irradiation. Furthermore, OE podocytes showed downregulated levels of NOX1 and NOX4 after RT. On the other hand, treatment with the NOX inhibitor GKT improved WTs' survival post-RT and restored SMPDL3b to basal levels. in vivo, the administration of GKT restored glomerular morphology and decreased proteinuria in 26-weeks irradiated mice. Taken together, these results suggest a novel role for NOX-derived reactive oxygen species (ROS) upstream of SMPDL3b in modulating the response of renal podocytes to radiation.

Phosphorus deficiencies invoke optimal allocation of exoenzymes by ectomycorrhizas

Ectomycorrhizal (EM) fungi can acquire phosphorus (P) through the production of extracellular hydrolytic enzymes (exoenzymes), but it is unclear as to the manner and extent native EM fungal communities respond to declining soil P availability. We examined the activity of six exoenzymes (xylosidase, N-acetyl glucosaminidase, β-glucosidase, acid phosphomonoesterase, acid phosphodiesterase [APD], laccase) from EM roots of Pseudotsuga menzesii across a soil podzolization gradient of coastal British Columbia. We found that APD activity increased fourfold in a curvilinear association with declining inorganic P. Exoenzyme activity was not related to organic P content, but at a finer resolution using 31P-NMR, there was a strong positive relationship between APD activity and the ratio of phosphodiesters to orthophosphate of surface organic horizons (forest floors). Substantial increases (two- to fivefold) in most exoenzymes were aligned with declining foliar P concentrations of P. menzesii, but responses were statistically better in relation to foliar nitrogen (N):P ratios. EM fungal species with consistently high production of key exoenzymes were exclusive to Podzol plots. Phosphorus deficiencies in relation to N limitations may provide the best predictor of exoenzyme investment, reflecting an optimal allocation strategy for EM fungi. Resource constraints contribute to species turnover and the assembly of distinct, well-adapted EM fungal communities.

Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities.

Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5′ exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA’s activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface β-loop that coordinates a fourth manganese ion (M4).

Purification and partial characterization of NAD aminohydrolase from Aspergillus oryzae NRRL447

Aspergillus oryzae aminohydrolase free acid phosphodiesterase catalyzes nicotinamide adenine dinucleotide to deamino-NAD and ammonia. The enzyme was purified to homogeneity by a combination of acetone precipitation, anion exchange chromatography and gel filtration chromatography. The enzyme was purified 230.5 fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of MW 94 kDa. The enzyme displayed maximum activity at pH 5 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable up to 40 °C. The enzyme activity was enhanced slightly by addition of Na⁺ and K⁺, whereas inhibited strongly by addition of Ag⁺, Mn²⁺, Hg²⁺ and Cu²⁺ to the reaction mixtures. The enzyme hydrolyzes several substrates, suggesting a probable non-specific nature. The enzyme catalyzes the hydrolytic cleavage of amino group of NAD, adenosine, AMP, CMP, GMP, adenosine, cytidine and cytosine to the corresponding nucleotides, nucleosides or bases and ammonia. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km and Kcat for the tested substrates were calculated.

Phosphatases and phosphodiesterases isolated from the red king crab (Paralithodes camtschatica) hepatopancreas

Five enzymes have been isolated from the hepatopancreas of the red king crab Paralithodes camtschatica by means of ion exchange and gel chromatography: two acid (AcP) and one alkaline (AlkP) phosphmonoesterases, one alkaline phosphodiesterase (AlkPD), and one acid phosphodiesterase (AcPD). The pH optimum values of these enzymes are: AlkPs and AlkPD, 7.5; AcP, 5.5; and AcPD, 5.0. The activity of AlkP and AlkPD demands Mg2+ ions. The molecular weights of the enzymes (kDa) are the following: AlkP, 80: AcPs, 80 and 82; AlkPD, 51; and AcPD, 57. The enzymes are relatively thermostable (ID50 from 47 to 62 degrees C). AlkP is inhibited by NaCl (IC50 at 0.4 M). The AcP, AcPD, and AlkPD activities are tolerant of high ionic strength.

The degradation of sodiumO,O‐diethyl dithiophosphate by bacteria from metalworking fluids

The breakdown of sodium O,O-diethyl dithiophosphate (O,O-diethyl phosphorodithioate) by four bacterial strains (tentatively identified as strains of Aeromonas, Pseudomonas, Flavobacterium and Bacillus) isolated from contaminated metalworking fluids was shown to involve the successive formation of ethanol, aldehyde and orthophosphate. An acid phosphodiesterase was identified in cell-free extracts that was five- to sevenfold enhanced in specific activity in bacteria grown on O,O-diethyl dithiophosphate as sole phosphorus source, compared with bacteria grown on orthophosphate. This is thought to initiate the breakdown process.

Purification and Characterization of Lupus Anticoagulant Like Protein from Agkistrodon Halys Brevicaudus Venom

By means of CM-Sephadex C-25, DEAE-Sephadex A-50, Sephadex G-200, and Sephadex G-75 chromatographies, a lupus anticoagulant like protein (LALP) from Agkistrodon halys brevicaudus was purified. On SDS-PAGE, the purified LALP had a molecular weight of 25,500 daltons under non-reducing condition and 15,000 daltons under reducing condition. The isoelectric point was pH 5.6. Its N terminal amino acid sequencing revealed a mixture of 2 sequences: DCP(P/S)(D/G)WSSYEGH(C/R)(Q/K). It was devoid of phospholipase A, fibrino(geno)lytic, 5'-nucleotidase, L-amino acid oxidase, phosphomonoesterase, phosphodiesterase and thrombin-like activities, which were found in crude venom. In the presence of LALP, PT, aPTT, and dRVVT of human plasma were markedly prolonged and its effects were concentration-dependent but time-independent. The inhibitory effect of LALP on the plasma clotting time was enhanced by decreasing phospholipid concentration in TTI test. The individual clotting factor activity was not affected by LALP when higher dilutions of LALP-plasma mixture were used for assay. Russell's viper venom time was shortened when high phospholipid confirmatory reagent was used. Therefore, the protein has lupus anticoagulant property.

Cellulose biosynthesis and function in bacteria

The current model of cellulose biogenesis in plants, as well as bacteria, holds that the membranous cellulose synthase complex polymerizes glucose moieties from UDP-Glc into beta-1,4-glucan chains which give rise to rigid crystalline fibrils upon extrusion at the outer surface of the cell. The distinct arrangement and degree of association of the polymerizing enzyme units presumably govern extracellular chain assembly in addition to the pattern and width of cellulose fibril deposition. Most evident for Acetobacter xylinum, polymerization and assembly appear to be tightly coupled. To date, only bacteria have been effectively studied at the biochemical and genetic levels. In A. xylinum, the cellulose synthase, composed of at least two structurally similar but functionally distinct subunits, is subject to a multicomponent regulatory system. Regulation is based on the novel nucleotide cyclic diguanylic acid, a positive allosteric effector, and the regulatory enzymes maintaining its intracellular turnover: diguanylate cyclase and Ca2(+)-sensitive bis-(3',5')-cyclic diguanylic acid (c-di-GMP) phosphodiesterase. Four genes have been isolated from A. xylinum which constitute the operon for cellulose synthesis. The second gene encodes the catalytic subunit of cellulose synthase; the functions of the other three gene products are still unknown. Exclusively an extracellular product, bacterial cellulose appears to fulfill diverse biological roles within the natural habitat, conferring mechanical, chemical, and physiological protection in A. xylinum and Sarcina ventriculi or facilitating cell adhesion during symbiotic or infectious interactions in Rhizobium and Agrobacterium species. A. xylinum is proving to be most amenable for industrial purposes, allowing the unique features of bacterial cellulose to be exploited for novel product applications.

Cellulose biosynthesis and function in bacteria.

The current model of cellulose biogenesis in plants, as well as bacteria, holds that the membranous cellulose synthase complex polymerizes glucose moieties from UDP-Glc into beta-1,4-glucan chains which give rise to rigid crystalline fibrils upon extrusion at the outer surface of the cell. The distinct arrangement and degree of association of the polymerizing enzyme units presumably govern extracellular chain assembly in addition to the pattern and width of cellulose fibril deposition. Most evident for Acetobacter xylinum, polymerization and assembly appear to be tightly coupled. To date, only bacteria have been effectively studied at the biochemical and genetic levels. In A. xylinum, the cellulose synthase, composed of at least two structurally similar but functionally distinct subunits, is subject to a multicomponent regulatory system. Regulation is based on the novel nucleotide cyclic diguanylic acid, a positive allosteric effector, and the regulatory enzymes maintaining its intracellular turnover: diguanylate cyclase and Ca2(+)-sensitive bis-(3',5')-cyclic diguanylic acid (c-di-GMP) phosphodiesterase. Four genes have been isolated from A. xylinum which constitute the operon for cellulose synthesis. The second gene encodes the catalytic subunit of cellulose synthase; the functions of the other three gene products are still unknown. Exclusively an extracellular product, bacterial cellulose appears to fulfill diverse biological roles within the natural habitat, conferring mechanical, chemical, and physiological protection in A. xylinum and Sarcina ventriculi or facilitating cell adhesion during symbiotic or infectious interactions in Rhizobium and Agrobacterium species. A. xylinum is proving to be most amenable for industrial purposes, allowing the unique features of bacterial cellulose to be exploited for novel product applications.

Characterization and partial purification of a novel mannosylphosphodolichol phosphodiesterase from chicken liver microsomes.

Mannosylphosphodolichol phosphodiesterase, which catalyzes the release of mannose from mannosylphosphodolichol, was solubilized from chicken liver microsomes by treatment with the non-ionic detergent, Emulgen 909. The enzyme was partially purified using ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration on Sepharose 6B. The enzyme showed absolute requirement for sulfhydryl reducing agents. The enzyme activity was stimulated by the addition of CaCl2 and Emulgen 909 and exhibited a pH optimum around 5.3. The Km value for mannosylphosphodolichol was found to be 0.43 microM. The activity was competitively inhibited by dolichyl phosphate and dolichol and the Ki value for dolichyl phosphate was estimated to be 12.5 microM. The purified preparation had no activity toward N-acetylglucosaminyldiphosphodolichol, glucosylphosphodolichol, mannose 1-phosphate, or artificial substrates for mannosidases, glucosidases, acid phosphatase, and acid phosphodiesterase. A heat-stable factor which stabilizes the mannosylphosphodolichol phosphodiesterase was separated from the enzyme by DEAE-cellulose chromatography. It was precipitated by trichloroacetic acid and not extracted into lipid solvents. The separation resulted in the complete loss of the enzyme activity and the restoration of the activity was not observed when the factor was added back to the enzyme solution.

Acid hydrolases during Artemia development: A role in yolk degradation

1. 1. A broad group of acid hydrolytic activities have been determined during Artemia development including cathepsin B acid ribonuclease, acid deoxyribonuclease, acid phosphatase, acid phosphodiesterase, β-glucosidase, β- N-acetylgalactosaminidase and acid lipase. These enzymes present maximum activity in nauplii, when yolk degradation is maximum. 2. 2. Artemia cathepsin B proteinase is able to degrade lipovitellin in vitro with a pattern similar to that found in vivo. These results suggest the involvement of acid hydrolases in the degradation of yolk. 3. 3. In cryptobiotic embryos, acid hydrolases were found associated with structures of buoyant density 1.18. The structures appeared at the electron microscope as 0.4 μm dia. vesicles when revealed by acid phosphatase cytochemistry.

Free-flow electrophoresis of the low-density structures that contain asialoglycoproteins in the rat liver.

Rats were given an intravenous dose (1-2 micrograms/100 g) of iodine-labelled asialotransferrin, asialofetuin, or asialoorosomucoid either alone or in combinations, and the distribution of the radioactivity in the liver, removed 10-20 min after the injection, was analyzed by free-flow electrophoresis in an Elphor VaP 11 apparatus. Liver homogenates were prepared for electrophoresis according to an elaborate ultracentrifugation scheme that is outlined in detail with respect to conditions and yields. The scheme involved differential centrifugation, followed by density gradient centrifugation in a linear sucrose gradient and gel filtration using Sepharose 2B. Two ligand-containing fractions were obtained during differential centrifugation, each associated with a different complement of subcellular marker enzymes. On free-flow electrophoresis, the ligand present in either fraction exhibited a major and a minor peak. They were incompletely separated, the minor peak shouldering on the major one. The major peak had a higher electrophoretic mobility than the peaks of the acid phosphatase and phosphodiesterase I activities, but it had the same mobility as the sialyltransferase activity. The minor, less electronegative peak comigrated with the peaks of acid phosphatase and phosphodiesterase I activities and also with the major protein component of the subcellular fraction. It is concluded that the asialoglycoprotein-transporting subcellular vesicles are heterogeneous in regard to charge and that their complete separation from subcellular marker enzymes cannot be accomplished by free-flow electrophoresis.

Cyclic AMP phosphodiesterases of Funaria hygrometrica

High affinity cAMP phosphodiesterase (PDE) activity has been purified ( ca 474-fold) from the chloronema cells of the moss Funaria hygrometrica. The activity in the 15 000 g supernatant was precipitated at 40–80% saturation with (NH 4) 2SO 4 and separated by DEAE-cellulose chromatography into two peaks of activity A and B with pH optima at pH 5.5 and 7.5, respectively. When rechromatographed on DEAE-cellulose, the PDE B activity redistributed into two peaks (A′and B′). The recovery of PDE activity in the latter peaks suggests a conversion of alkaline PDE into acid PDE. The phosphodiesterase activity B was further resolved by affinity chromatography on a cAMP—agarose column into PDE I and PDE II. The latter was free from nucleotidases, was Ca 2+-dependent and showed only a high-affinity component towards cAMP as the substrate, the K m being 11.8 ± 4.8 μM.

Development and physiology of infection by the downy mildew fungus Peronospora parasitica (Pers. ex Fr.) Fr. in susceptible and resistant Brassica species

The growth of two isolates of the downy mildew fungus Peronospora parasitica, one obtained from cauliflower (Brassica oleracea) and the other from oilseed rape (B. napus) was assessed in their respective hosts of origin, and also in the alternative combination. Both isolates were capable of infecting either host, but there were marked contrasts in the time course and extent of mycelial development, the amounts of associated host‐cell necrosis, and eventual intensity of sporulation. Oilseed rape, which was partially resistant to the isolate from cauliflower, exhibited extensive necrosis of mesophyll cells, in conjunction with reduced mycelial development, and delayed and reduced sporulation by the pathogen. The isolate from oilseed rape was virulent on both host species. Pathogenesis in the susceptible combinations was accompanied by large increases in electrolyte leakage, and increased activity of the enzymes β‐glucosidase, ribonuclease, and peroxidase. Effects on chlorophyll content were variable and activities of acid phosphatase and acid phosphodiesterase were unaffected. Electrophoretic analyses of extracts from fungal sporangia and infected seedlings indicated that the large increases in β‐glucosidase were of pathogen origin, while evidence from inhibitor studies suggested that enhanced ribonuclease activity was due to a new post‐infectional form of the enzyme. The significance of these findings is discussed in relation to pathogenesis and host resistance mechanisms.

Subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinases and phosphoprotein phosphatase in Trypanosoma brucei

The subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinases and phosphoprotein phosphatase in bloodstream forms of Trypanosoma brucei was determined by isopycnic sucrose-gradient centrifugation of post-large-granule extracts. Cyclic-AMP phosphodiesterase was almost entirely soluble whereas adenylate cyclase was membrane-bound. The latter enzyme appeared to be absent from the plasma-membrane fraction but copurified with acid phosphatase and acid phosphodiesterase indicating a possible association with the flagellar pocket. At least two protein kinase activities could be distinguished as based on their distribution profiles in gradients, their preference for exogenously added acceptor protein and their inhibition and stimulation by suramin and nucleoside, respectively. Suramin-sensitive protein kinase co-purified with the plasma-membrane marker α-D-glucosidase and a nucleoside-stimulated protein kinase behaved as a typical cell-sap enzyme. Phosphoprotein phosphatase activity was found to be mainly soluble but a small part seemed to be associated with plasma membranes.

Localization of hydrolases in cultured procyclic trypomastigotes of Trypanosoma brucei

Procyclic culture forms of Trypanosoma brucei were examined for enzymes that could serve as markers for the plasma membrane, flagellar pocket, lysosomes, endoplasmic reticulum and Golgi apparatus in order to study the subcellular organization of the digestive system of the parasite. By analogy with a previous investigation on the parent bloodstream forms of the same parasite strain, acid phosphatase, acid phosphodiesterase, alanine aminotransferase, galactosyltransferase, α-glucosidase, inosine diphosphatase, α-mannosidase and acid proteinase were selected. With the exception of acid proteinase, α-mannosidase and neutral α-glucosidase, the specific activities of the enzymes studied were significantly lower in procyclic than in bloodstream forms. The degree of association of the selected hydrolases with particulate material as well as their structural latency were determined. Cell fractionation experiments revealed that both α-mannosidase and acid proteinase co-banded with particles at a density of about 1.16 g/cm 3. Inosine diphosphatase, α-glucosidase and acid phosphatase sedimented mainly as microsomal constituents and equilibrated at densities between 1.13 and 1.15 g/cm 3 in sucrose gradients. Though predominantly soluble, part of the alanine aminotransferase was membrane-associated. In cytoplasmic extracts, acid phosphatase, inosine diphosphatase, acid proteinase and α-mannosidase were particle-bound and latent, whereas α-glucosidase was completely accessible to its substrate. In intact cells five of the six enzymes tested exhibited high degrees of latency, including soluble alanine aminotransferase, a marker of cell integrity, as well as α-glucosidase and acid phosphatase. Only acid phosphodiesterase showed a substrate accessibility of 44%. Except for alanine aminotransferase the enzyme activities in cell-free extracts were highly sedimentable (70–100%). Pre-incubation with 0.1% Triton X-100 markedly reduced the sedimentability of the enzyme activities, with the exception of inosine diphosphatase which appeared to be strongly bound to membranes. It is tentatively concluded that (a) acid proteinase and α-mannosidase are located in lysosomes; (b) acid phosphatase is associated with microsomal membranes derived from endoplasmic reticulum and digestive tubules and that, in contrast to the situation in the bloodstream form, it is absent from the flagellar pocket; (c) only trace amounts of acid phosphodiesterase are present in the flagellar pocket; and (d) α-glucosidase may be a constituent of the plasma membrane.

A lysosomal enzyme involved in diphosphatidylglycerol degradation.

A soluble lysosomal phosphodiesterase in rat liver that hydrolyzes monoacylglycerophosphorylglycerophosphorylglycerol (AGPGPGase) was shown to be distinct from a lysosomal acid phosphodiesterase IV (PDase IV) which catalyzes the hydrolysis of bis(p-nitrophenyl) phosphate. The criteria used to distinguish lysosomal AGPGPGase from PDase IV were: separation on ion exchange celluloses, dissimilar inhibition patterns and different rates of inactivation on concentration. The lysosomal PDase IV activity was competitively inhibited by inorganic phosphate with a Ki value of 0.33 mM phosphate and was inhibited by a number of organophosphoryl compounds including AGPGPG, phosphatidylcholine, phosphatidylinositol, ATP and 4-methylumbelliferylpyrophosphate.

Acid deoxyribonuclease, acid phosphodiesterase and acid phosphatase of new born rat epidermis (skin): Multiple forms and glycoprotein nature

1. Isoelectric focusing and concanavalin A-Sepharose were used to study multiple forms and glycoprotein nature of acid phosphohydrolases from new born rat epidermis (skin). 2. The crude extracts were focused in a 110 ml Ampholine gradient column with pH ranges of 3.5–10, 7–9 and 5–7. Acid DNase was resolved into three different forms, acid phosphodiesterase and acid phosphatase were separated into two different forms. All these enzymes separated by electrophoresis showed the optimal pH at 5.0. 3. The bulk of acid DNase and acid phosphodiesterase were bound to the column of concanavalin A-Sepharose, whereas about 30% of acid phosphatase passed through. About 50% of acid phosphatase were eluted with -methyl- d-glucoside. About 22% of acid DNase and about 42% of acid phosphodiesterase were released from the column with α-methyl- d-glucoside. These results indicate glycoprotein nature of these acid hydrolases.

Isopycnic-zonal centrifugation of plasma membrane, sarcoplasmic reticular fragments, lysosomes, and cytoplasmic proteins from phasic skeletal muscle

Homogenates of the posterior latissimus dorsi muscle, a phasic muscle, were fractionated by a one-step zonal centrifugation technique into four major organelle populations and cytoplasmic constituents. These were: (1) Plasma membrane fragments with a modal equilibrium density of 1.10 and containing 5′-nucleotidase, alkaline phosphodiesterase, p- nitrophenylphosphatase and acid phosphatase (β-glycerophosphate was used as the substrate). (2) Sarcoplasmic reticular fragments which could be further subdivided into calcium transport vesicles, with a modal equilibrium density of 1.16, that exhibited calcium uptake; K +-ATPase; leucyl-β-naphthylamidase; acid phosphodiesterase; acid phosphatase (using cytidine monophosphate as the substrate); and sarcoplasmic reticular lysosomes, with a modal equilibrium density of 1.18, possessing dipeptidyl-aminopeptidase II, cathepsin D, α-glucosidase, N- acetyl-β- glucosaminidase , and NADH oxidase activity. (3) Mitochondria with a modal equilibrium density of 1.21. (4) Catalase-containing vesicles with a modal equilibrium density of 1.22; and cytoplasmic constituents (modal density of 1.25) with phosphorylase, pyruvate kinase, myosin-ATPase, aldolase, and protein and RNA content. The purity of these organelles was equal to or better than previous efforts, with a 30-fold purification achieved for 5′-nucleotidase and alkaline phosphodiesterase. These results lend support to the hypothesis that the sarcoplasmic reticulum of phasic muscle, in addition to its specialized role in excitation-contraction coupling, represents a multifunctional membrane system, and that, similar to the smooth endoplasmic reticulum of other cells, it includes some membrane-bound lysosomal enzymes and NADH oxidase.

Purification and characterization of acid phosphodiesterases of cultured tobacco cells.

Two acid phosphodiesterases (phosphoric diester hydrolase, E.C. 3.1.4) were purified to apparent homogeneity from cultured cells of Nicotiana tabacum var. Bright Yellow. These enzymes appeared to differ in some physicochemical properties but exhibited similar broad substrate specificities towards cyclic and p-nitrophenyl nucleotides, and bis-p-nitrophenyl phosphate. Of these compounds, the most active substrates were bis-p-nitrophenylphosphate and 2',3'-cyclic nucleotides. Both p-nitrophenyl 3'- and 5'-thymidylates were hydrolyzed at comparable rates, whereas the hydrolysis of 3',5'-cyclic AMP occurred preferentially at the 5'-phosphodiester bond. A unique catalytic property of these acid phosphodiesterases was the ability to liberate inorganic phosphate from ATP, ADP and p-nitrophenyl phosphate. Ribonucleic acid, its breakdown products, and inorganic phosphate were found to be effective as inhibitors.