Halina Sierakowska

Continuous fluorimetric assay of nucleotide pyrophosphatase. Kinetics, inhibitors, and extension to dinucleoside oligophosphatases

Abstract A simple and convenient procedure is described for the continuous fluorimetric assay of nucleotide pyrophosphatase (dinucleotide nucleotidohydrolase, EC 3.6.1.9) activity. It is based on the multifold increase in fluorescence emission intensity accompanying hydrolysis of a new fluorogenic substrate, ϵAp 2 ϵA (where ϵ = 1, N 6 -etheno), or of commercially available ϵNAD + and FAD, using potato nucleotide pyrophosphatase. The procedure is applicable to enzyme activity in tissue extracts, as well as to kinetic studies and screening of potential inhibitors. K m and V max / K m for the substrates, and K i values for various compounds, were evaluated. The most effective inhibitor of ϵAp 2 ϵA hydrolysis was the alternative substrate 8-bromo-NAD + ( K i = 7 μ M). The method is also useful for continuous assay of snake venom phosphodiesterase I-5′-exo-nuclease (EC 3.1.4.1). The preparation of ϵAp 2 ϵA is described, and spectral properties of the fluorogenic substrates listed. Both ϵAp 3 ϵA and ϵAp 4 ϵA were also shown to be fluorogenic substrates for nucleotide pyrophosphatase and phosphodiesterase I, and may be used for continuous fluorimetric assay of specific dinucleoside oligophosphatases.

Mammalian Nucleolytic Enzymes and Their Localization

Publisher Summary This chapter describes the mammalian nucleolytic enzymes and their localization. A multitude of the enzymes of varying degrees of specificity exhibits hydrolytic activity against ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and nucleoside cyclic phosphates. Attempts at the direct intracellular localization of nucleolytic enzymes have been confined largely to the differentiated materials of higher organisms. Pancreatic RNase is most abundant in the pancreas, but similar activity is found in other tissues and body fluids. A ribonuclease with specificity similar to that for the pancreatic enzyme has been isolated and purified 260-fold from KB cultured mammalian epithelial cells. DNase I was first crystallized from the pancreas, but similar activity occurs in other organs. The enzyme is optimally active at about pH 7 and is usually activated by Mg 2+ . Native DNA is depolymerized, with a rapid decrease in viscosity, and denatured DNA is attacked more slowly. The enzyme is inactive toward small oligonucleotides, esters of mononucleotides, apurinic acid, and deaminated single-stranded DNA.

Purification and resolution of potato tuber cyclic nucleotide phosphodiesterase from nucleotide pyrophosphatase

Abstract Cyclic nucleotide phosphodiesterase from potato tubers has been purified 8000-fold to near homogeneity in standard SDS-polyacrylamide gel electrophoresis. Apart from previously described activities against nucleoside 2′,3′- and 3′,5′-(cyclic) phosphates, bis( p -nitrophenylphosphate) and p -nitrophenylphosphate, the purified enzyme cleaved aryl esters of nucleoside 3′- and 5′-phosphates, aryl phosphonates, nucleoside di- and triphosphates and the 2′,3′-GMP terminal residue of a fragment of TMV RNA. It was inactive against pyrophosphate linkages in NAD + , m 7 GpppGm and ADP[βS] and the phosphodiester linkages in A-A, cAMP[S] and arabinosylytosine 2′,5′-(cyclic)phosphate. Relative rates of activity, and K m values for several substrates, were determined. Particular significance is attached to the removal from the enzyme preparation of virtually all activity against internucleotide pyrophosphate linkages, hitherto considered an inherent property of both cyclic nucleotide phosphodiesterase and nucleotide pyrophosphatase. These two enzymes with identical M r were found to separate in SDS-gel electrophoresis without prior reduction. Potential contamination of other plant cyclic nucleotide phosphodiesterase with nucleotide pyrophosphatase and consequent ability to cleave the 5′-terminal m 7 GpppGm of mRNA were reassessed. Cyclic nucleotide phosphodiesterase was found to occur in monomeric and oligomeric forms, and conditions for their interconversion were established. Both forms exhibited similar rates of activity against various substrates. Gel filtration and sucrose density gradient centrifugation led to an M r of 79000–81000 for the monomer and 343000–346000 for the oligomer. The Stokes radii were 35 A and 58 A, respectively, subunit M r of 74000 was determined by SDS-gel electrophoresis.

A specific colorimetric and cytochemical substrate for ribonuclease T2: Adenosine-3′-(α-naphthylphosphate)

Abstract Details are presented for the synthesis of adenosine-3′-(α-naphthylphosphate), a specific substrate for the cytochemical localization or colorimetric assay of ribonuclease T 2 (ribonucleate nucleotido-2′-transferase (cyclizing), EC 2.7.7.17) type enzymes. The synthetic route involves the preparation of 3′-O- acetyladenosine , which is converted stepwise to N 6 - benzoyl-2′,5′-di -O- tetrahydropyranyladenosine , followed by phosphorylation of the latter with either α-naphthylphosphoryl dichloride, or with α-naphthylphosphate in the presence of dicyclohexylcarbodiimide. Final purification of the product was attained by column chromatography on benzyl-DEAE cellulose, a procedure superior to those previously employed for purification of other nucleoside-3′-(α-naphthylphosphate) substrates. Comparative rates of hydrolysis for this substrate by ribonuclease T 2 , ribonuclease T 1 (EC 2.7.7.26), pancreatic ribonuclease (EC 2.7.7.16) and phosphodiesterase II (EC 3.1.4.1) are presented and compared with those for other substrates of these enzymes. The overall findings are evaluated in terms of substrate specificities and their possible overlapping.

A ribonuclease substrate resistant to phosphodiesterase II: 5′-O-benzyluridine-3′-(α-naphthylphosphate)

Summary A study has been made of the influence of various 5′-substituents on the susceptibility to hydrolysis by pancreatic ribonuclease, ribonuclease T 2 , and phosphodiesterase II, of uridine-3′-( α -naphthyl-phosphate). It was established that 5′-O-benzyluridine-3′-( α -naphthylphosphate) is a suitable ribonuclease substrate for cytochemical and colorimetric purposes, and fully resistant to phosphodiesterase II. The synthesis of this substrate is described.

Reassociable dimer subunit of potato nucleotide pyrophosphatase; specificity and stability

Abstract Treatment of the tetramer ( M r 340 000) of nucleotide pyrophosphatase (dinucleotide nucleotidohydrolase, EC 3.6.1.9) from potato tubers with glycine-NaOH buffer (pH 10) for 1–2 h leads to its dissociation to an active dimer with an M r , estimated from sucrose density gradient centrifugation and modified SDS-polyacrylamide gradient gel electrophoresis, of 170 000. In acidic (pH ≈ 5), and to some extent in neutral media the dimer reassociates to the tetramer. The activity of the dimer is considerably less stable than that of the tetramer. The specific activities of the dimer and tetramer towards thymidine 5′- p -nitrophenylphosphate are identical, whereas the rates of cleavage of pyro- and oligophosphates derived from 5′-nucleotides, such as dT(5′)p 2 (5′)dT, Ap 2 A, Ap 3 A and NAD + , are up to 70% lower for the dimer. By contrast, for substrates derived from 3′-nucleotides such as thymidine 3′- p -nitrophenylphosphate, 2′,3′-cAMP and dT(3′)p 2 (3′)dT as well as for bis( p -nitrophenyl)phosphate and p -nitrophenylphenylphosphonate, the dimer subunit is 3–10-fold more active than tetramer. These increases in activity are not accompanied by large changes in K m values. A modified SDS-polyacrylamide gradient gel electrophoresis technique, adapted for the foregoing study, should be equally applicable to determination of M r values of other oligomeric proteins, especially enzymatically active ones.