Steady-state kinetic studies of the inhibitory action of Zn2+ on ribonuclease T1 catalysis.

The kinetic mechanism of specific inhibition by Zn2+ of ribonuclease T1 catalysis was studied by steady-state kinetic analysis of transphosphorylation of dinucleotides, GpCp(3'), GpUp(2') and GpUp(3'), and dinucleoside monophosphates, GpC and GpU. The inhibition was not simply competitive, non-competitive or uncompetitive, but the kinetic data were compatible with a mechanism of 'fully mixed inhibition' in which a fully non-competitive action was associated with a partially competitive action. Apparent equilibrium quotients involved in this model of inhibition were determined for the dinucleotide substrates, and we found that binding of either of Zn2+ and substrate was facilitated when the other was bound. The location of Zn2+ was suggested to be near His-40 and/or His-92 of the ribonuclease T1 molecule.     

Zinc(II)-mediated inhibition of ribonuclease Sa by an N-hydroxyurea nucleotide and its basis

Ribonuclease Sa (RNase Sa) is a secretory ribonuclease from Streptomyces aureofaciens. Herein, 3'-N-hydroxyurea-3'-deoxythymidine 5'-phosphate is shown to be a competitive inhibitor of catalysis by RNase Sa. Inhibition is enhanced by nearly 10-fold in the presence of Zn(2+), which could coordinate to the N-hydroxyurea group along with enzymic residues. The carboxylate of Glu54 is the putative base that abstracts a proton from the 2' hydroxyl group during catalysis of RNA cleavage by RNase Sa. Replacing Glu54 with a glutamine residue has no effect on the affinity of N-hydroxyurea 1 for the enzyme, but eliminates the zinc(II)-dependence of that affinity. These data indicate that an N-hydroxyurea nucleotide can recruit Zn(2+) to inhibit the enzymatic activity of RNase Sa, and suggest that the carboxylate of Glu54 is a ligand for that Zn(2+). These findings further the development of a new class of ribonuclease inhibitors based on the complex of an N-hydroxyurea nucleotide and zinc(II).     

Effect of nucleic-acid-binding compounds on the hydrolytic activity of various ribonucleases.

Studies were conducted on the stimulatory effect that various nucleic-acid-binding compounds have on the hydrolysis of RNA and polyribonucleotides by pancreatic ribonuclease A and by other ribonucleases. The stimulatory activity of chloroquine on tRNA hydrolysis by pancreatic ribonuclease was due to the formation of oligonucleotides of a wide range of sizes and was not due to the formation of very short ( n greater than 5) oligonucleotide fragments of tRNA. The dextrorotatory and levorotatory isomers of chloroquine did not differ in their ability to stimulate the hydrolysis of tRNA by pancreatic ribonuclease A. In addition to chloroquine and primaquine, other nucleic-acid-binding compounds (e.g., quinacrine, lucanthone, and proflavin) stimulated the hydrolysis of tRNA by pancreatic ribonuclease A. Chloroquine did not alter the rate of hydrolysis by pancreatic ribonuclease A of low-molecular-weight substrates (cytidine cyclic 2':o'-monophosphate, uridine cyclic 2':3'-monophosphate, cytidylyl-adenosine, or uridylyl-uridine). Furthermore, chloroquine and primaquine did not affect the hydrolysis of poly(A) by high concentrations of pancreatic ribonuclease A. In studies on the hydrolysis of tRNA by other endoribonucleases, several of the nucleic-acid-binding compounds (e.g., quinacrine and ethidium) exhibited appreciable inhibition of both ribonuclease N1 and ribonuclease T1. None of the compounds tested stimulated the activity of ribonuclease T1, and only chloroquine, and perhaps lucanthone, stimulated the hydrolysis of tRNA by ribonuclease N1.     

A ribonuclease from yeast associated with the 40 S ribosomal subunit.

1. Autodegradation of yeast ribosomes is due to a 'latent' ribonuclease which is associated with the 40 S ribosomal subunit. 2. The ribonuclease was extracted in the presence of EDTA from ribosomes and purified 118-rold by protamine sulphate precipitation, (NH4)2SO4 fractionation and chromatography on DEAE-cellulose. 3. The optimum pH for this enzyme is 5 to 6.5 while the optimum temperature is 45 to 50 degrees C. Incubation for 10 min at 60 degrees C caused a reduction in enzyme activity of 70%. 4. The ribonuclease has an endonucleolytic activity against rRNA, tRNA, poly(A), poly(U) and poly(C) but does not degrade poly(G) or DNA. It hydrolyzes the homopolymers to nucleoside 3'-phosphates. 5. Zn2+, Mn2+, heparin, glutathione and p-chloromercuribenzoate inhibit the ribonuclease, while Na+, K+, EDTA and sermidine have only little or no effect. 6. It binds tightly to yeast ribosomes but only loosely to ribonuclease-free wheat germ ribosomes. 7. Polyribosomes possess less autodegradation activity than monoribosomes, isolated from the same homogenate.     

Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn2+ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn2+ ions in catalysis. Whereas Zn2+ destabilizes substrate ground state binding to an extent that precludes reliable Kd determination, Co(NH3)6(3+) and Sr2+ in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn2+ and Co(NH3)6(3+) substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr2+ on the ribozyme reaction with Zn2+ as cofactor could be rationalized by a model involving two Sr2+ ions (or two classes of Sr2+ ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn2+. A single 2'-fluoro modification at nt -1 of the substrate substantially weakened the inhibitory effect of Sr2+. Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg2+ entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E*S formation apart from the catalytic performance.     

Characterization of the Extracellular Ribonuclease of Tetrahymena pyriformis W

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.     

A ribonuclease from human skeletal muscle

1. A ribonuclease has been prepared from human muscle by ammonium sulphate fractionation, heat treatment and ion-exchange chromatography. 2. The enzyme degrades polycytidylic acid and polyuridylic acid to the nucleoside 3'-phosphates, with nucleoside 2':3'-cyclic phosphates as intermediates. Polyadenylic acid and polyguanylic acid are not attacked. 3. The enzyme has maximal activity at pH8.5. The molecular weight (by gel filtration) is between 11000 and 12000. It is relatively heat-stable. It exhibits optimum activity in a medium of high ionic strength, and is inhibited by several bivalent cations, particularly Zn(2+).     

Characterization of the extracellular ribonuclease of Tetrahymena pyriformis W

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.     

Effect of zinc(II) and other divalent cations on binding of 3,5,3'-triiodo-L-thyronine to nuclear receptors from cultured GC cells

The effect of Zn(II) in 3,5,3'-triiodo-L-thyronine (T3) binding to nuclear receptors was studied in dialyzed 0.4 M NaCl extracts of nuclei from cultured GC cells. Addition of ZnCl2 to nuclear extracts resulted in a time- and concentration-dependent dissociation of T3 from nuclear receptors. Half-maximal dissociation occurred at 6 microM ZnCl2. Addition of ZnCl2 also resulted in a concentration-dependent inhibition of binding of T3 to nuclear receptors. Half-maximal inhibition of binding occurred at 1-3 microM ZnCl2. Scatchard analysis indicated that Zn(II) addition decreased kA and did not alter receptor concentration. These effects of Zn(II) were prevented when ZnCl2 was added to nuclear extracts in the presence of 5 mM EDTA or 5 mM dithiothreitol. Moreover, Zn(II)-induced inhibition of T3 binding was reversed by the addition of 5 mM EDTA. The inhibitory effect of Zn(II) on T3 binding seemed specific for nuclear receptors; no effect of Zn(II) on the binding of T3 to proteins in rat serum or GC cell cytosol or to rabbit anti-T3 serum was observed. Cd(II) had a similar concentration-dependent inhibition of T3 binding to nuclear receptors which was reversible. Our findings suggest that Zn(II) may play a role in T3 binding to nuclear receptors as well as its putative role in the binding of receptor to DNA.     

Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease

Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.     

The role of zinc ions in the transformation of lymphocytes by phytohaemagglutinin

1. Incorporation of [(3)H]thymidine into DNA was inhibited by EDTA and diethylenetriamine-NNN'N'N'-penta-acetate but not by nitrilotriacetate even when Ca(2+) was present at more than twice the concentration of the chelators. 2. The inhibition caused by EDTA was most effectively reversed by Zn(2+) but also to a lesser extent by Cd(2+). Very little if any activation of the EDTA-inhibited system was obtained with Co(2+), Cu(2+), Fe(3+), Mn(2+) or Ni(2+) added alone. 3. Fe(3+) added to the Zn(2+)-activated lymphocytes in the presence of EDTA markedly increased thymidine incorporation. Addition of Cd(2+) prevented the inhibition of incorporation which occurred at high Zn(2+) concentrations. 4. If EDTA was added more than 15h after phytohaemagglutinin, the inhibition of incorporation was less than that obtained by its addition at zero time. If Zn(2+) was added later than 12h after EDTA and phytohaemagglutinin, the inhibition of incorporation by EDTA was not fully reversed. A study of the time-course of the effects of delayed additions of EDTA and Zn(2+) suggested that, on average, the cells required Zn(2+) between 20 and 30h after phytohaemagglutinin addition to allow the full rate of thymidine incorporation at 37h. 5. The increase in the rate of protein synthesis caused by phytohaemagglutinin was not inhibited by EDTA until about 8h. The further increase after this was totally inhibited by EDTA but this inhibition was fully reversible with Zn(2+). The rate of protein synthesis in EDTA-inhibited cultures at 40h was the same as that at 10h. 6. There was a close similarity between the effects of EDTA on lymphocyte stimulation and those reported by Kay et al. (1969) with low doses of actinomycin D.     

Ribonuclease inhibitor from human placenta: rapid purification and assay.

The ribonuclease inhibitor from human placenta may be isolated in 65% yield (2.5 mg per placenta) in 2 days. The performance of the affinity chromatography on Sepharose-RNase A has been expedited through adaption of the spectrophotometric assay of ribonuclease toward 2',3'-cyclic cytidine monophosphate to determination of the inhibitor activity. The result of these improvements in procedure is increased availability of the ribonuclease inhibitor for study of its chemical properties (Blackburn, P., and Jailkhani, B.L. (1979) J. Biol. Chem. 254, 12488-12493) and for its inclusion into in vitro systems in which inhibition of mammalian neutral ribonucleases is desired.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Extracellular ribonuclease formation in Bacillus subtilis and its stimulation by actinomycin D

1. Extracellular ribonuclease is produced linearly for at least 3hr. by washed post-logarithmic-phase cells of Bacillus subtilis suspended in a medium containing maltose (1%) and casein hydrolysate (0·5%). 2. Low concentrations of actinomycin D (less than 2μg./ml.) stimulate ribonuclease formation, the maximum effect being observed with a concentration of 1μg./ml. Concentrations greater than 2μg./ml. are inhibitory. There is no parallel stimulation of α-amylase formed under the same conditions, and [¹⁴C]uracil incorporation into a perchloric acid-insoluble form is inhibited. 3. The actinomycin D-induced stimulation is not due to the presence of an activator, nor is the inhibition due to the release of an inhibitor by the cells. The effect is on the amount of ribonuclease produced in the medium. 4. Extracellular ribonuclease formation is partially inhibited by anaerobiosis, 2,4-dinitrophenol, sodium azide and by chloramphenicol and puromycin. 5. High concentrations of antibiotic do not completely inhibit ribonuclease formation, but a basal amount of enzyme representing 20min. synthesis in an uninhibited system is always produced. This `antibiotic-insensitive' enzyme could possibly represent preformed enzyme `in the pipe-line' en route to secretion. 6. The stimulated appearance of ribonuclease in the presence of 1μg. of actinomycin D/ml. is shown to be dependent on enzyme synthesis. The mechanism of this effect is discussed.     

The role of zinc ions in reverse transport mediated by monoamine transporters

The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.     

Green tea polyphenols as inhibitors of ribonuclease A

Ribonucleases (RNases), which are essential for cleavage of RNA, may be cytotoxic due to undesired cleavage of RNA in the cell. The quest for small molecule inhibitors of members of the ribonuclease superfamily has become indispensable with a growing number exhibiting unusual biological properties. Thus, inhibitors of RNases may serve as potential drug candidates. Green tea catechins (GTC), particularly its major constituent (-)-epigallocatechin-3-gallate (EGCG), have reported potential against cell proliferation and angiogenesis induced by several growth factors including angiogenin, a member of the RNase superfamily. This study reports the inhibition of bovine pancreatic ribonuclease A (RNase A) by EGCG and GTC. This has been checked qualitatively by an agarose gel based assay. Enzyme kinetic studies with cytidine 2',3' cyclic monophosphate as the substrate have also been conducted. Results indicate substantial inhibitory activity of a noncompetitive nature with an inhibition constant of approximately 80 microM for EGCG and approximately 100 microM for GTC measured in gallic acid equivalents.     

Ribonuclease inhibitors in Malus x domestica (common apple): isolation and partial characterization

A ribonuclease inhibitory activity was detected in the fruits of common apple, Malus x domestica, cv. Fuji, and purified by affinity chromatography on ribonuclease A-Sepharose. It inhibited hydrolysis of cyclic-2':3'-CMP by bovine pancreatic ribonuclease A with an apparent inhibition constant of about 5 x 10(-8) M. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the purified protein gave two peaks corresponding to the mass numbers of 55,658 and 62,839, while three bands of 43-, 34-, and 21-kDa were detected by SDS-PAGE. These results suggested that the inhibitor preparation was a mixture of two proteins comprised of 43- and 21-kDa subunits or of 34- and 21-kDa subunits. Attempts to separate these two proteins were unsuccessful. Amino acid composition and N-terminal amino acid sequence of these subunits were also identified and N-terminal sequences showed some similarity to that of cottonseed storage globulin. The significance of the presence of ribonuclease inhibitors in apple fruits is not clear, but it might allow some speculation about their possible involvement in the control of the self-incompatibility ribonuclease of Rosaceae plants.     

Germ Tube Growth Inhibitor from Cronartium comandrae Aeciospores

The reduction of nucleic acid by an endogenous polynucleotide phosphorylase and ribonuclease in cells of Brevibacterium JM98A (ATCC 29895) was studied. A simple process was developed for the activation of the endogenous RNA-degrading enzyme(s). RNA degradation was activated by the presence of Pi with 14.2 mumol of ribonucleoside 5'-monophosphate per g of cell mass accumulating extracellularly. The optimum pH for degradation of RNA was 10.5 and the optimum temperature was 55 to 60 degrees C. Enzymatic activity was inhibited by the presence of Ca2+, Zn2+, or Mg2+. Although some of the RNA-degrading enzymatic activity was associated with the ribosomal fraction, most was soluble. Both polynucleotide phosphorylase and ribonuclease activities were identified.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.