Probes of eukaryotic DNA-dependent RNA polymerase II-I. Binding of 9-beta-D-arabinofuranosyl-6-mercaptopurine to the elongation subsite

9-beta-D-Arabinofuranosyl-6-mercaptopurine (ara-6-MP) was used to affinity-label wheat germ DNA-dependent RNA polymerase II (or B) (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). This nucleoside analogue was found to be a competitive inhibitor with respect to [3H]UMP incorporation. Natural substrates protected the enzyme from inactivation by ara-6-MP when the enzyme was preincubated with excess concentrations of substrates, suggesting that the inhibitor binds at the elongation subsite. The inhibitor bound the catalytic center of the enzyme with a stoichiometry of 0.6:1. The sulfhydryl reagent, dithiothreitol, reversed the inhibition by ara-6-MP, suggesting that the 6-thiol group of the inhibitor was interacting closely with an essential cysteine residue in the catalytic center of the enzyme. Chromatographic analysis of the pronase-digestion products of the RNA polymerase II-ara-6-MP complex also showed that ara-6-MP had bound a cysteine residue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured [6-35S]ara-6-MP-labeled RNA polymerase II revealed that over 80% of the radioactivity was associated with the IIb subunit of the enzyme.     

The binding of sulfonamides to horse liver alcohol dehydrogenase

The binding of sulfonamides to the active site of horse liver alcohol dehydrogenase has been studied by their effect on affinity labelling and steady state kinetics. Affinity labelling with iodoacetate and BIP has been used to study binding to free enzyme. The unsubstituted sulfonamide, sulfanilamide (I), shows very weak binding compared to the other sulfonamides tested. Most important for binding is the type of substituent attached to the parent sulfonamide, particularly when as in sulfathiazole this is a heterocycle which binds to the catalytic zinc atom of the enzyme. For sulfathiazole the dissociation constant from the enzyme is pH dependent showing two pK_a values. The lower at pH 7 is the pK_a of the drug itself, while that at pH 9 agrees with the ionization of water bound to the catalytic zinc ion.Steady state kinetics have been carried out at pH 7.0 and 10.0 to examine sulfonamide binding to the enzyme when coenzyme is attached. Both NAD⁺ and NADH induce substrate competitive sulfonamide binding. Likewise sulfathiazole accelerates the dissociation of NADH from the enzyme and so V_max for alcohol oxidation. The latter like stimulation of the affinity labelling reaction with iodoacetate is considered to result from binding of the thiazole ring to the catalytic zinc ion. With all the sulfonamides examined hydrophobic binding and charge are important in determining affinity to the active site and the mode of binding. Sulfonamides containing pyrazole or imidazole rings can be important in alcohol therapy.     

Hepatic macromolecular binding following exposure to vinyl chloride

Covalent binding of radioactivity to hepatic macromolecules in rats exposed to ¹⁴C-labeled vinyl chloride (VC) was studied to determine if VC-induced carcinogenesis may be related to electrophilic alkylation of macromolecules in vivo. Male Sprague-Dawley rats were exposed to 1, 10, 25, 50, 100, 250, 500, 1000, or 5000 ppm of [¹⁴C]VC for 6 hr. Following exposure, radioactivity covalently bound to hepatic macromolecules and purified nucleic acids (RNA, DNA) was determined. The total amount of [¹⁴C]VC metabolized and hepatic glutathione (GSH) content were also determined. The total amount of radioactivity bound to macromolecules in the liver did not increase proportionately to the increase in the exposure concentration of VC. A disproportionate decrease in macromolecular binding was observed as the concentration of VC increased. The covalent binding to hepatic macromolecules was related to the amount of VC metabolized. At exposures greater than 50 ppm, the amount of ¹⁴C bound to macromolecules in the liver correlates with induction of hepatic angiosarcoma. There was no detectable binding of radioactivity to either DNA or RNA in the liver. Hepatic glutathione content was significantly depressed only at exposure concentrations greater than 100 ppm.     

Biochemical effects of d-tetrandrine and thalicarpine.

The benzylisoquinoline alkaloids d-tetrandrine and thalicarpine inhibit the biosynthesis of DNA, RNA and proteins, when incubated with S180 cells in vitro. Oxidation of glucose[¹⁴C] to ¹⁴CO₂ was not affected by either alkaloid at levels up to 100 μg/ml in vitro. Incorporation of labeled acetate into lipids was inhibited only by thalicarpine at 100 μg/ml. Inhibition of the incorporation of thymidine into DNA was also observed in vivo after treatment with these drugs at 30–120 mg/kg; under these conditions, the synthesis of RNA and protein was not inhibited. In an attempt to elucidate the mechanism for inhibition of nucleic acid synthesis, the interaction of DNA, RNA and polynucleotides with the alkaloids was studied by gel filtration and dialysis. The two drugs associated with both DNA and RNA, but exhibited different affinities for the five polynucleotides examined. Both alkaloids were bound by polyguanylic and polyadenylic acids, but whereas d-tetrandrine associated only poorly with polythymidylic acid and not at all with polyuridylic acid, it was polycytidylic acid that showed no affinity for thalicarpine.     

Inhibition of hypoxanthine-guanine phosphoribosyl transferase.

The affinities of eighteen purines or purine analogs for human erythrocytic hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase) were compared to assess the feasibility of obtaining active inhibitors of the enzyme. Three compounds appeared to inhibit the utilization of hypoxanthine by L5178Y cells in vitro due to inhibition of the enzyme rather than depletion of the intracellular 5-phosphoribosyl-1-pyrophosphate pool. The three competitive inhibitors and their affinity constants (K_i) using 6-mercaptopurine as substrate were: 6-mercapto-9-(tetrahydro-2-furyl)-purine, 37 μM; 2,6-bis-(hydroxyamino)-9-β-d-ribofuranosyl-purine, 12 μM; and 6-iodo-9-(tetrahydro-2-furyl)-purine, 108 μM. The KInm for 6-mercaptopurine was 9 μM. Thus, the enzyme tolerates bulky substitution at N⁹. 6-Mercapto-9-(tetrahydro-2-furyl)-purine also potentiated the chemotherapeutic effect of azaserine, an inhibitor of de novo purine biosynthesis, in L5178Y ascites tumor-bearing mice. Four 2-substituted, oxazolo-[5, 4-d]-pyrimidine-7-ones and 2-methylthiazolo-[5, 4-d]-pyrimidine-7-one had K_i values in the range of 84–173 μM. Consequently, isosteric substitution at N⁹ may also be a fruitful and logical course to pursue in the design and synthesis of more potent inhibitors of this important enzyme.     

Synthesis,characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6–22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase

PKI(6–22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (K_i= 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine¹⁰)PKI(6–22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-³H]phenylalanine¹⁰)PKI(6–22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and U.V. spectra. In the dark, (4-azidophenylalanine¹⁰)PKI(6–22)amide inhibited the catalytic subunit of CAMP-dependent protein kinase with a K_i value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent U.V. spectral changes on exposure to light. Photolysis of the catalytic subunit.(4-azido[3,5-³H]phenylalanine¹⁰)PKI(6–22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.     

Regiospecific arylazo substitution into α-amanitin with retention of inhibitory properties against eukaryotic Class II RNA polymerase

Treatment of α-amanitin with 1,2 equiv. benzenediazonium acetate in methanolic NaOH affords the crude 7'-phenylazo-α-amanitin in 92% yield. The azoamatoxin is isolable in yields of 50% following reverse phase partition chromatography and shows the extended conjugation of an azo dye (Λ_max= 295 and 384 nm in CH₃OH) when compared to the parent α-amanitin (Λ_max= 305 nm). Spectrophotometric titration of the amatoxin phenolic hydroxyl shows a decrease in pK_A to 7.75 as expected following azo substitution. Proton magnetic resonance at 60 MHz shows 7 aromatic protons; two AB coupled protons (δ_4H= 7.83 p.p.m.; δ_5H= 6.80 p.p.m., J_4,5= 9) are assigned to the remaining two protons of the amatoxin indole. The 5 phenyl protons constitute a second order spin system with δ_3,4,5H= 7.44–7.53 p.p.m. and δ_2,6H= 7.97–8.11 p.p.m. The 7'-phenylazo-α-amanitin inhibits sea urchin RNA polymerase with apparent K_is at 1.4 times 10^-8M (class II) and 1.3 times 10^-5M (class III). The K_i for the urchin class III RNA polymerase is decreased 10-fold from the value observed with the parent α-amanitin (K_i= 1.4 times 10^-4M), while there is no significant change for the class II enzyme. No inhibition of the class I or E. coli RNA polymerase is observed at 10^-4M. Extension of this diazo coupling reaction using other arylamines affords the preparation of a family of 7'-arylazo-α-amanitins in good yield analogous to the benzenediazonium reaction. The title reaction thus provides a second facile, efficient route to modified amatoxins of defined structure with retention or enhancement of inhibitory activity against the eukaryotic RNA polymerase.     

AFM study of Escherichia coli RNA polymerase σ⁷⁰ subunit aggregation

The self-assembly of Escherichia coli RNA polymerase σ⁷⁰ subunit was investigated using several experimental approaches. A novel rodlike shape was reported for σ⁷⁰ subunit aggregates. Atomic force microscopy reveals that these aggregates, or σ⁷⁰ polymers, have a straight rodlike shape 5.4 nm in diameter and up to 300 nm in length. Atomic force microscopy data, Congo red binding assay, and sodium dodecyl sulfate gel electrophoresis confirm the amyloid nature of observed aggregates. The process of formation of rodlike structures proceeds spontaneously under nearly physiological conditions. E. coli RNA polymerase σ⁷⁰ subunit may be an interesting object for investigation of amyloidosis as well as for biotechnological applications that exploit self-assembled bionanostructures. Polymerization of σ⁷⁰ subunit may be a competitive process with its three-dimensional crystallization and association with core RNA polymerase.From the Clinical EditorIn this basic science study, the self-assembly of Escherichia coli RNA polymerase σ⁷⁰ subunit was investigated using atomic force microscopy and other complementary approaches.     

Posttranslationally modified ornithine decarboxylase may regulate RNA polymerase I activity

Purified ornithine decarboxylase (EC 4.1.1.17, ODC) transamidated with four putrescine moieties on four glutamine residues through the action of transglutaminase (EC 2.3.2.13, TGase) purified from guinea pig liver, when added to isolated rat liver nuclei, stoichiometrically increased the activity of RNA polymerase I (EC 2.7.7.6). Te increase was relative to the pmoles of purified conjugated ODC added to the reaction and could be reinitiated after the reaction had plateaued by the further addition of ODC-putrescine conjugate. The kinetics of the reaction suggest that the ODC-putrescine conjugate was not reused but degraded after each initiation. Otherwise, the rapid plateau would not be observed. The repeated addition of 278 pmoles of purified ODC-putrescine conjugate to rat liver nuclear preparations containing 200 μg total protein consistently stimulated the incorporation of 600–700 pmoles UMP/mg protein. We suggest that ODC transmidated by its product putrescine may be the posttranslationally modified 65,000 M_r protein which has been reported by several laboratories to serve as a labile subunit of RNA polymerase I.     

Inhibition of methylation of nuclear ribonucleic acid in L1210 cells by tubercidin, 8-azaadenosine and formycin

The adenosine analogs tubercidin (7-deazaadenosine), formycin (7-amino-3-[β-d-ribofuranosyl] pyrazolo[4,3-d]pyrimidine) and 8-azaadenosine were examined for their effects on the synthesis and methylation of nuclear RNA in L1210 cells in vitro. Total RNA and DNA synthesis was affected to the greatest extent by tubercidin (IC₅₀ = 7 × 10^?6M) and to an insignificant degree by 8-azaadenosine and formycin; however, the effects of the latter two drugs, but not of tubercidin, were potentiated by 2'-deoxycoformycin, an inhibitor of adenosine deaminase. In the presence of 2'-deoxycoformycin, RNA synthesis was inhibited by 40 per cent at 1 × 10^?4 M 8-azaadenosine and by 50 per cent at 2 × 10^?4 M formycin, while DNA synthesis was inhibited less extensively. Alkaline hydrolysis of nuclear RNA labeled with [¹⁴C]uridine and l-[methyl-³H]methionine showed preferential inhibition of base methylation in mononucleotides, but not of 2′-O-methylation in dinucleotides, for all three drugs. This differential effect persisted to varying degrees in ?18S and 4S nuclear RNA separated by electrophoresis. The reduction in base methylation in 4S RNA was associated with seven of the eight methylated nucleosides in 4S RNA separated by two-dimensional thin-layer chromatography. These results indicate that tubercidin, 8-azaadenosine and formycin can preferentially inhibit the base methylation of nuclear RNA relative to its synthesis.     

Synthesis of RNA in the pineal gland during N-acetyltransferase induction

The biosynthesis of RNA in the cultured rat pineal was monitored during the (l)-isoproterenol-induced increase in N-acetyltransferase activity. Treatment with the β-agonist (l)-isoproterenol elevated pineal N-acetyltransferase activity 30-fold within 6 hr but had no effect on the incorporation of [³H]uridine into total nuclear, total cytoplasmic or total poly(A) containing RNA. Examination of the [³H]uridine-labeled total cytoplasmic RNA and poly(A)-rich messenger RNA by means of agarose gel electrophoresis revealed no significant changes in the synthesis of these RNA species by (l)-isoproterenol treatment of the cultured pineal gland. The lag in the appearance of newly synthesized RNA from the nucleus into the cytosol of the pineal closely corresponds to the lag in the appearance of new N-acetyltransferase activity. These results suggest that, although (l)-isoproterenol may cause a dramatic induction of N-acetyltransferase enzyme activity in the cultured pineal gland, there is no significant alteration in the RNA biosynthesis of the pineal gland to meet the demands of enzyme induction. The induction of N-acetyltransferase activity by (l)-isoproterenol is inhibited by actinomycin D. However, sensitivity to the antibiotic is lost 90–180 min after the initiation of induction by (l)-isoproterenol but 90 min before the accumulation of appreciable enzyme activity.     

Structure-toxicity relationships in the amatoxin series Synthesis of S-deoxy[γ(R)-hydroxy-Ile3]-amaninamide,its crystal and molecular structure and inhibitory efficiency*§

The amatoxins, highly toxic components of Amanita mushrooms, strongly inhibit the DNA-dependent RNA polymerase II (or B) in eukaryotic cell nuclei. For optimal binding to the enzyme a γ-hydroxyisoleu-cine side chain in the 3-position is important as in γ-amanitin (compound l), where the OH-group is bound in the [S]-configuration. Amanullin, a non-toxic component, having an oxygen-free isoleucine side chain no. 3, exhibits an inhibitory effect on RNA polymerase II about two orders of magnitude smaller than that of γ-amanitin. An equal, relatively weak, inhibitory effect has previously been found with the synthetically obtained Ile³-analog 7. In the present paper the synthesis of an analog (2) bearing a γ-hydroxyl group in the isoleucine side chain is described. The compound was found to have about the same inhibitory effect on RNA polymerase II from Drosophila embryos as amanullin and the Ile³-analog 7. Structure analysis by X-ray diffraction revealed that the hydroxyl group at the -carbon atom of side chain-3 has the [R]-configuration, the new analog thus being -deoxo[()-hydroxy-[Ile³]-amaninamide. It follows that the [S]-configuration of this chiral center is a prerequisite to maximal toxicity. Crystallographic data demonstrating great similarity between the peptide backbones of the new analog and those of natural amatoxins are given.     

3H]Ouabain binding and dissociation in rabbit colon: effect of ions and drugs

²²Na fluxes (J) and [³H]ouabain binding were studied in vitro in the distal portion of the rabbit colon, mounted as a membrane, separating two perspex chambers. J_MS^Na (flux of ²²Na from mucosal to serosal chamber) was 2.1 and J_SM^Na was 1.0 μmoles cm² × hr· J_SM^Na was completely abolished by 10^?3 M ouabain, placed in the serosal side. J_SM^Na was unaffected by ouabain. [³H] Ouabain binding to the serosal surface of the colon was inhibited to the same maximal effect by ‘cold’ ouabain (I₅₀ = 5 × 10^?7 M), by K⁺ placed in the serosal chamber (I₅₀ = 3.5 mM) and by replacement of Na⁺ with choline on the serosal side. Increased tissue concentration of Na⁺ did not affect [³H]ouabain binding, suggesting that extracellular rather than intracellular sodium plays a major role in cardiac glycoside binding. Various cardiac glycosides (digoxin, digitoxin. ouabain) inhibited [³H]ouabain binding to the serosal side of the colon but other steroids (estriol, testosterone, desoxycorticosterone) had not effect.Efflux of [³H]ouabain, bound to the serosal side of the colon, was differentiated into dissociation of nonspecifically bound [³H]ouabain (95 per cent in 60 min) and dissociation of specifically bound [³H]ouabain (20 per cent in 60 min). Dissociation of specifically bound [³H]ouabain was accelerated when Na was replaced by choline (50 per cent in 60 min). The dissociation rate of nonspecifically bound ouabain was unaffected by replacement of Na⁺· [³H]Ouabain binding to mucosal surface was unaffected by ‘cold’ ouabain, by increased K⁺ in the medium or by replacement of Na⁺. It is concluded that there is no specific binding of [³H]ouabain to the mucosal surface. Omission of Ca, Mg or phosphate from the medium did not affect [³H]Ouabain binding to either mucosal or serosal surfaces of rabbit colon.     

Synthesis and pharmacological characterization of [I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74 ± 0.97 nM. At a concentration equivalent to its K_d, non-specific binding comprised 27 ± 2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR (t_1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity (K_d, nM) to recombinant A₃ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.     

Studies on the inhibition of poly(A) polymerase from rat liver and hepatoma 3924A by rifamycin SV derivatives

The inhibition of poly (A) polymerase activity from liver and hepatoma 3924A by several O-n-alky derivatives of rifamycin SV:3-formyloxime was studied. Only the O-n-pentyl (AF/012) and the O-n-octyl (AF/013) analogs were active at the maximum tested concentration of 1 × 10^?4 M. Equivalent concentrations of liver and hepatoma enzymes were inhibited to the same degree by both compounds. The 50 per cent inhibitory concentration (IC₅₀) for AF/013 and AF/012 was 1.2 × 10^?5M and 7.5 × 10^?5M respectively. Poly (A) polymerase activity was more sensitive to AF/013 if the enzyme was preincubated with the drug before the initiation of the reaction with poly (A) than if the reaction was initiated by the addition of the enzyme. Addition of AF/013 after initiation of the assay led to a rapid inhibition of poly (A) synthesis. The mechanism of inhibition by AF/012 and AF/013 of poly(A) polymerase was of the non-competitive type with respect to ATP.     

Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.

RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.     

High-throughput screening identification of poliovirus RNA-dependent RNA polymerase inhibitors

Viral RNA-dependent RNA polymerase (RdRP) enzymes are essential for the replication of positive-strand RNA viruses and established targets for the development of selective antiviral therapeutics. In this work we have carried out a high-throughput screen of 154,267 compounds to identify poliovirus polymerase inhibitors using a fluorescence based RNA elongation assay. Screening and subsequent validation experiments using kinetic methods and RNA product analysis resulted in the identification of seven inhibitors that affect the RNA binding, initiation, or elongation activity of the polymerase. X-ray crystallography data show clear density for five of the compounds in the active site of the poliovirus polymerase elongation complex. The inhibitors occupy the NTP binding site by stacking on the priming nucleotide and interacting with the templating base, yet competition studies show fairly weak IC₅₀ values in the low μM range. A comparison with nucleotide bound structures suggests that weak binding is likely due to the lack of a triphosphate group on the inhibitors. Consequently, the inhibitors are primarily effective at blocking polymerase initiation and do not effectively compete with NTP binding during processive elongation. These findings are discussed in the context of the polymerase elongation complex structure and allosteric control of the viral RdRP catalytic cycle.     

Hydrolysis of opioid hexapeptides by carboxypeptidase N: Presence of carboxypeptidase in cell membranes

Carboxypeptidase N, purified to homogeneity from human plasma, rapidly hydrolyzed Lys⁶-or Arg⁶-enkephalins when measured by high pressure liquid chromatography. Comparison of the kinetics of hydrolysis of the enkephalin hexapeptides and bradykinin by carboxypeptidase N revealed the following values for the K_m and K_cat: Arg⁶-Met⁵-enkephalin, 49 μM, 1024 min^?1; Arg⁶-Leu⁵-enkephalin, 57 μM, 375 min^?1; Lys⁶-Met⁵-enkephalin, 216 μM, 6204 min^?1; bradykinin, 19 μM, 58 min^?1. Thus, while bradykinin had the lowest K_m, the specificity constants (K_cat/K_m) for all the enkephlain hexapeptides were higher than that of bradykinin due to their high turnover numbers. Preincubation of the enzyme with 0.1 mM CoCl₂ increased both the K_cat and K_m of bradykinin and Arg⁶-Met⁵-enkephalin. Similar results were obtained when the above experiments were conducted with the active 48,000 dalton subunit of carboxypeptidase N. Basic carboxypeptidase activity was found in the amniotic fluid, in membrane fractions of various human and bovine tissues, and in cultured cells in the following order of decreasing specific activity: human placental muvilli, human kidney, human amniotic fluid, human lung, bovine lung, bovine pulmonary artery, human foreskin fibroblasts, human pulmonary arterial endothelial cells, and human lung fibroblasts. The membrane-bound carboxypeptidase activity had a neutral pH optimum and behaved similarly to plasma carboxypeptidase N in the presence of various inhibitors and activators. It was different from the carboxypeptidase activity in bovine adrenal chromaffin granules which had an acid pH optimum and was inhibited by sulfhydryl reagents. These studies show that human carboxypeptidase N, an enzyme found in high concentration in blood, readily hydrolyzes Arg⁶- or Lys⁶-enkephalins. It could thus control the levels of these peptides if they are released into the circulation from the adrenal gland. In addition, a membrane-bound carboxypeptidase N-like enzyme in various tissues may regulate the local levels of biologically active peptides containing C-terminal basic amino acids such as hexapeptide enkephalins, kinins, anaphylatoxins or fibrinopeptides.