Saccharomyces cerevisiae DNA-dependent RNA polymerase III: a zinc metalloenzyme.

Yeast nuclear RNA polymerase III was purified by batch adsorption to phosphocellulose, followed by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on DNA-Sepharose. Polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band which contained polymerase activity. The molecular weight estimated by sedimentation velocity centrifugation in a glycerol gradient was 380 000. Enzyme activity was inhibited 50% at 0.1 mM 1,10-phenanthroline and 100% of 1.0 mM, but was restored when 1,10-phenanthroline was removed by dialysis. Enzyme activity was not inhibited by 7,8-benzoquinoline, a nonchelating structural analogue of 1,10-phenanthroline. These results strongly suggest that inhibition of enzyme activity occurs by the formation of a reversible enzyme-zinc-phenanthroline ternary complex. The zinc content, measured by atomic absorption spectroscopy, was 2 g-atoms per mol of enzyme. Zinc was not removed from the enzyme by gel filtration on Sephadex G-25, by passage through Chelex-100 resin, or by dialysis against buffer containing 1,10-phenanthroline. Enzyme-bound zinc was removed by dialysis after denaturation of the enzyme with heat and sodium dodecyl sulfate. Enzyme-bound zinc did not exchange with free zinc. These results establish yeast nuclear RNA polymerase III as a zinc metalloenzyme.     

Leukotriene A4 hydrolase: a zinc metalloenzyme

Purified human leukotriene A4 hydrolase is shown to contain 1 mol of zinc per mol of enzyme, as determined by atomic absorption spectrometry. The enzyme is inhibited dose-dependently by the chelating agents 8-hydroxy-quinoline-5-sulfonic acid, and 1,10-phenanthroline with KI values of about 2 and 8 x 10(-4) M, respectively, whereas dipicolinic acid and EDTA are ineffective in this respect. The inhibition by 1,10-phenanthroline is time-dependent, and at a concentration of 5 mM, 50% inhibition of enzyme (3 x 10(-7) M) occurs after about 15 min. The zinc atom of leukotriene A4 hydrolase can be removed by dialysis against 1,10-phenanthroline which results in loss of enzyme activity. The catalytic activity is almost completely restored by the addition of stoichiometric amounts of Zn2+ or Co2+.     

The role of Zn(II) in transcription by T7 RNA polymerase

Homogeneous T7 RNA polymerase contains from 2–4 gm atoms of zinc per mole of M.W. 107,000. Inactivated molecules which can be separated from the active molecules by repeated chromatography contain less zinc, from 0.4 to 1 gm at per mole. Instability of the enzyme makes it difficult to relate maximal activity to a specific stoichiometry of Zn. The enzyme is inhibited by 1,10-phenanthroline, EDTA, CN^?, SH^?, N₃^? and by incubation with Chelex resin. Zinc is retained on gel filtration, but can be removed by dialysis for 96 hr against 5 mM 1,10-phenanthroline which totally inactivates the enzyme. Catalytic activity requires the presence of thiol reagents. Preparations with low activity can be activated by exogenous Zn ions.     

The zinc-containing high Km cyclic nucleotide phosphodiesterase of bakers' yeast

The high Km cyclic nucleotide phosphodiesterase of Saccharomyces cerevisiae was purified by an improved procedure. Its amino acid composition is reported. Its pI is 5.85 +/- 0.1. Sedimentation equilibrium analysis of the native enzyme gave Mr = 88,000 +/- 6,000, whilst gel electrophoresis in the presence of dodecyl sulfate gave a molecular weight of 43,000, indicating that the enzyme is a dimer. Preparations of 94 +/- 4% purity contained about 2.4 atoms of zinc/43,000 daltons. Inactivation of the enzyme by 8-hydroxyquinoline was accompanied by removal of about 2 zinc atoms per monomer. Partially inactivated enzyme regained activity during dialysis against zinc, or, with less effect, cobalt salts. 8-Hydroxyquinoline (Ki = 1.1 mM) and 1,10-phenanthroline (Ki = 0.6 mM) were competitive inhibitors. The enzyme was also inhibited by the nonchelating 1,7-and 4,7-phenanthrolines and by thiols and KCN, but not by NaN3. These inhibitors probably act by binding to, but not chelating, enzyme-bound zinc.     

State and accessibility of zinic in yeast alcohol dehydrogenase.

1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited in the presence of 1,10-phenanthroline. 2. A conformational change in the enzyme's structure is induced by 1,10-phenanthroline, and is abolished in the presence of NADH. 1,10-Phenanthroline binds to the enzyme competitively with respect to NADH, with a stoicheiometry of 2 mol of 1,10-phenanthroline/144000g of enzyme. 3. 1,10-Phenanthroline induces a time-dependent dissociation of Zn2+ from the enzyme, which is in correlation with its inhibitions. 4. Spectrophotometric measurement indicates that the dissociation of half (2 zinc atoms/tetramer) of the total zinc content of the enzyme correlates with the full inhibition of its activity. Measurement of the tightly bound Zn2+ by atomic absorption photometry confirms this. 5. A proposition is advanced that the tetrameric molecule of yeast alcohol dehydrogenase possesses an inherent asymmetry, with four monomeric subunits being arranged in two mutually symmetrical pairs.     

Inhibition of E. coli DNA polymerase I by 1,10-phenanthroline.

A 1,10-phenanthroline-cuprous ion complex is a potent reversible inhibitor of $\text{E. coli}$ DNA polymerase I yielding 50% inhibition in the micromolar concentration range. The 2:1 1,10-phenanthroline-cuprous ion complex is most probably the inhibitory species. Complexes of cupric ion and 1,10-phenanthroline have no apparent kinetic effect. The previously reported inhibition of the enzyme by 1,10-phenanthroline (1,2) is most likely due to the formation of this complex from thiols normally added to the assay mixtures and trace amounts of cupric ion invariably present notwithstanding reasonable precaution. The reversible and instantaneous 1,10-phenanthroline inhibition observed for other polymerases may be due to this unique inhibitory species and not coordination of a catalytically important zinc ion at the active site by the chelating agent.     

Protein synthesis elongation factor 1 from rat liver: A zinc metalloenzyme

Highly purified elongation factor 1 (light form, EF1_L) from rat liver contains zinc as determined by atomic absorption spectrophotometry. Analysis has been performed on the most active protein fraction from DEAE-Sephadex chromatography (estimated purity: 90%) and on the main band obtained from this fraction by polyacrylamide gel electrophoresis. The data are consistent with a stoichiometry of approximately one g-atom of zinc per 54,000 daltons of EF1_L protein. A functional role for Zn²⁺ is suggested by the fact that 0.3 mM 1,10-phenanthroline completely abolishes GTP binding by EF1_L (measured by the nitrocellulose filter retention assay), while the isomeric non-chelator 1,7-phenanthroline has no effect. This inhibition can be overcome by the addition of excess zinc ion.     

Zinc metalloenzyme properties of active and latent collagenase from rabbit bone.

1. Inhibition of collagenase from rabbit bone cultures by the chelating agents 1,10-phenanthroline and EDTA is almost completely reversed by Zn2+; other metal cations are less effective in reversing the inhibition. Optimal restoration of activity is achieved at Zn2+ concentrations below that of the chelator, but excess of Zn2+ is inhibitory. 2. Prolonged incubation of collagenase with either chelator causes irreversible inactivation. This inactivation is prevented by Zn2+ at the same concentrations needed to reverse the primary inhibition. 3. Collagenase incorporates 65Zn by exchange when incubated with 1,10-phenanthroline and Zn2+ containing this radioactive isotope. The 65Zn2+ can be removed from its binding site in collagenase by 1,10-phenanthroline or EDTA. Irreversible inactivation of collagenase by chelators destroys its ability to incorporate 65Zn2+. 4. Latent collagenase, the inhibited form in which collagenase first appears in culture, behaves similarly to the active enzyme in 65Zn2+-exchange experiments, but is resistant to irreversible inactivation by chelators. 5. It is concluded that collagenase is a zinc metalloenzyme that forms an inactive and unstable apoenzyme on treatment with chelators. The bound inhibitor component of latent collagenase evidently stabilizes the apoenzyme.     

Limiting rates of ligand association to alcohol dehydrogenase.

Detailed stopped-flow kinetic studies of the association of 2,2-bipyridine, 1,10-phenanthroline, and 5-chloro-1,10-phenanthroline to the zinc ion at the active site of alcohol dehydrogenase have demonstrated that a process with a limiting rate constant of about 200 s^?1 restricts the binding of the bidentate chelating agents to the free enzyme. The formation of the enzyme-ligand complexes has been followed by means of the characteristic absorption spectra of the resulting complexes or by the displacement of the fluorescent dye, auramine O. Monodentate ligands, upon binding to the free enzyme or enzyme-NAD⁺ and enzyme-NADH complexes, do not exhibit a comparable limiting rate. In analogy with simple inorganic systems, these observations have been interpreted in terms of the rate limiting dissociation of an inner sphere water molecule following the rapid formation by the bidentate ligand of an outer sphere complex. The displacement of a water molecule from the zinc ion by 1,10-phenanthroline has been observed in crystallographic studies which have also established that the zinc ion in the enzyme-1,10-phenanthroline complex is pentacoordinate. Monodentate ligands, which are substrate analogs, do not exhibit limiting rates because displacement of water is not required for their addition to a coordinate position which is apparently vacant in the free enzyme. If a water molecule remains bound to the zinc ion in the kinetically competent ternary complex, it could play an essential role in the proton transfer reaction accompanying catalysis.     

Enzymic synthesis of lignin precursors. Further studies on cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures

Isoenzyme 2 of cinnamyl-alcohol dehydrogenase from soybean suspension cultures was purified about 3800-fold to apparent homogeneity by an improved purification procedure involving biospecific elution of the enzyme from a NADP+-agarose column. On sodium dodecylsulfate gels the dehydrogenase showed only one protein band with Mr 40 000 +/- 500. The enzyme is strongly inhibited by thiol reagents. Various metal chelators as well as the nonchelating 7,8-benzoquinoline also inhibited enzyme activity. Inhibition by 10 mM 1,10-phenanthroline could be partially reversed by addition of Zn2+. 1,10-Phenanthroline and 7,8-benzoquinoline are non-competitive inhibitors with respect to NADP+. The presence of zinc in the dehydrogenase was proved by atomic absorption spectroscopy and by specific incorporation of 65Zn into the enzyme. In steady-state kinetics inhibition patterns were obtained which are consistent with an ordered bi-bi mechanism in which NADP(H) is the first substrate to bind and the last product released. The cinnamyl-alcohol dehydrogenase belongs to the A-specific dehydrogenases and removes the pro-R hydrogen from coniferyl alcohol. The enzyme shows many similarities with alcohol dehydrogenases from horse and rat liver and from yeast.     

Plasmodium berghei: effect of protease inhibitors during gametogenesis and early zygote development

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.     

The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80 degrees C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl2 was able to restore activity, and the binding stoichiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in kcat/Km values of 1-5 x 10(1) s(-1) mM(-1).     

1,10-Phenanthroline-cuprous ion complex, a potent inhibitor of DNA and RNA polymerases.

The inhibition by 1,10-phenanthroline of $\text{E. coli}$ DNA polymerase I has recently been attributed to the formation in the assay mixtures of a unique and effective inhibitor, the 2:1 1,10-phenanthroline-cuprous ion complex (1). We have now found that this coordination complex is also an effective inhibitor of $\text{E. coli}$ DNA dependent RNA polymerase, Micrococcus luteus DNA dependent DNA polymerase, and T-4 DNA dependent DNA polymerase. This conclusion is based either on the requirement of a thiol for 1,10-phenanthroline inhibition or on the reversal of 1,10-phenanthroline inhibition by the non-inhibitory cuprous ion specific chelating agent 2,9-dimethyl-1,10-phenanthroline. 2,2′,2″-Terpyridine is also very effective at relieving 1,10-phenanthroline inhibition. The reversal of 1,10-phenanthroline inhibition should be attempted before it is claimed that 1,10-phenanthroline inhibits any polymerases by coordinating a zinc ion at the active site.     

Affinity labelling of 5-aminolevulinic acid dehydratase with 2-bromo-3-(5-imidazolyl)propionic acid

2-Bromo-3-(5-imidazolyl)propionic acid, a zinc-directed thiol reagent, inactivates the enzyme 5-aminolevulinic acid dehydratase from bovine liver (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing, EC 4.2.1.24). The substrate, 5-aminolevulinic acid, completely protects against inactivation. The reagent inhibits the zinc-containing enzyme to a greater extent than the zinc-deprived enzyme; and it competes with the zinc chelator 1,10-phenanthroline. The reagent alkylates essential sulfhydryl groups of the enzyme, since the extent of the inactivation depends on the reduction of the enzyme protein by thiol compounds. It is concluded that the zinc site, the substrate site and the essential sulfhydryl groups are in close proximity in the active site.     

1,10-Phenanthroline phosphorylates (activates) MAP kinase in Xenopus oocytes

The membrane-permeable intracellular heavy metal chelator, 1,10-phenanthroline, which prevents progesterone-induced germinal vesicle breakdown (GVBD), would be expected to regulate phosphorylation (activation) of the MAP kinase (MAPK) cascade in Xenopus oocytes. Here, our experiments show that 1,10-phenanthroline itself results in the phosphorylation of MAPK in both oocytes and a cell-free system. In contrast, 1,7-phenanthroline, the nonchelating analogue, had no effect. A supplement of zinc (as a heavy metal) given to 1,10-phenanthroline-loaded oocytes suppressed the stimulatory effects of 1,10-phenanthroline, while 1,10-phenanthroline withdrawal caused dephosphorylation of activated MAPK. Further, treatment with a MEK (a MAPK kinase) inhibitor, PD 098059 or U0126, suppressed 1,10-phenanthroline-stimulated MAPK phosphorylation, indicating that 1,10-phenanthroline can phosphorylate MAPK in a MEK-dependent fashion. Our results suggest that phosphorylation of MAPK by 1,10-phenanthroline depends on the interaction of MEK. Thus, the intracellular heavy metal (zinc) regulates MAPK phosphorylation and 1,10-phenanthroline can serve as a unique tool for investigating MAPK phosphorylation mechanism.     

A study in situ of the effect of metallo- and serine proteinase inhibitors on the birefringence of the secretory stage enamel organic extracellular matrix

Abstract

Dental enamel formation occurs extracellularly and establishment of an ordered enamel organic extracellular matrix (ECM) seems to be crucial for proper construction of the enamel mineral phase. Polarizing microscopy shows that the ordered supramolecular structure of the secretory stage enamel organic ECM exhibits strong birefringence. We reported earlier that this birefringence is lost in unfixed specimens, probably due to extensive proteolytic cleavage of enamel proteins. Therefore, we investigated the association between enamel proteinase activities by analyzing the effects of metallo- and serine proteinase inhibitors in situ on the birefringence of the secretory stage enamel organic ECM. Male rats were used in the present study. After sacrifice, distal 10 mm fragments of upper incisors were removed and immersed for 15 h under continuous shaking at 37°C in one of the following solutions: 1) 10 mM Tris, pH 8.0; 150 mM NaCl (negative control, n = 8); 2) 2% paraformaldehyde and 0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2 (positive control, n = 5); 3) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline (n = 9); 4) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM phenylmethyl-sulfonyl fluoride (PMSF) (n = 8); 5) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline; 2 mM PMSF (n = 9). Samples then were immersed in fixative solution for 24 h and processed to obtain 5 μm thick longitudinal sections of the secretory stage enamel organic ECM. The sections were immersed in 80% glycerin for 30 min and analyzed by transmitted polarizing light microscopy. 1,10-Phenanthroline (inhibitor of metalloproteinases) and 1,10-phenanthroline + PMSF (inhibitor of serine proteinases) clearly prevented a decrease in the optical retardation of birefringence brightness from the tissue. PMSF alone promoted a slight preservation of the birefringence exhibited by the secretory stage enamel organic ECM. Rapid loss of birefringence in secretory stage enamel organic ECM that is not fixed immediately is caused by enamel proteinases and the activity of metalloproteinases seems to lead to preliminary degradation of the enamel organic ECM, which in turn facilitates subsequent serine proteinase activity.     

The carboxyl methyltransferase modifying G proteins is a metalloenzyme.

The prenylated protein carboxyl methyltransferase (PPMT) catalyzes the posttranslational methylation of isoprenylated C-terminal cysteine residues found in many signaling proteins such as the small monomeric G proteins and the gamma subunits of heterotrimeric G proteins. Here we report that both membrane-bound PPMT from rat kidney and the recombinant bacterially expressed form of the enzyme required divalent cations for catalytic activity. Unlike EDTA and EGTA, the metal chelator 1,10-phenanthroline strongly inhibited the PPMT activity of kidney intracellular membranes in a dose- and time-dependent manner. 1,10-Phenanthroline was found to inhibit the methylation of the prenylcysteine analog N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, a synthetic substrate for PPMT, with an IC(50) of 2.2 mM. Gel electrophoretic analysis demonstrated that 1,10-phenanthroline almost totally abolished the labeling of methylated proteins in kidney intracellular membranes. Immunoblotting analysis showed that one of the two major peaks of (3)H-methylated proteins in intracellular membranes comigrated with the small G proteins Ras, Cdc42, RhoA, and Rab1. In addition, the methylation of immunoprecipitated Ras and RhoA from kidney intracellular membranes was strongly inhibited when 1,10-phenanthroline was present. Treatment of kidney intracellular membranes with 1,10-phenanthroline increased the proteolytic degradation of PPMT by exogenous trypsin, compared to untreated membranes. We conclude from these data that metal ions are essential for the activity and the stabilization of PPMT. The finding that PPMT is a metalloenzyme may provide new insights into the functions played by this methyltransferase in signal transduction processes.     

A metalloproteinase from human rheumatoid synovial fibroblasts that digests connective tissue matrix components. Purification and characterization

Human rheumatoid synovial cells in culture stimulated with the conditioned culture medium of rabbit macrophages secrete three distinct latent metalloproteinases. One of them, a proteinase that digests proteoglycan and other connective tissue matrix components, was purified as two active forms after activation with 4-aminophenylmercuric acetate. The two forms were homogeneous on sodium dodecyl sulfate-gel electrophoresis with Mr = 45,000 and Mr = 28,000, whereas the latent precursor was estimated to have Mr = 51,000 by gel permeation chromatography. Both active enzymes had optimal activity at pH 7.5-7.8 and were inhibited by EDTA and 1,10-phenanthroline but not by inhibitors for cysteine, serine, or aspartic proteinases. Removal of Ca2+ from the enzyme solution resulted in a complete loss of activity that could be fully restored by the addition of 1 mM Ca2+. The activity of the apoenzyme was restored by the addition of 0.5 mM Zn2+, 5 mM Co2+, or 5 mM Mn2+ in the presence of Ca2+ but not by each metal ion alone. The identical digestion patterns of reduced, carboxymethylated protein substrates indicated that both active forms of the enzyme have the same substrate specificity. The enzyme degraded cartilage proteoglycans, type I gelatin, type IV collagen, laminin, and fibronectin, and removed the NH2-terminal propeptides from chick type I procollagen. This enzyme may play a role in the normal turnover of the connective tissue matrix as well as in the joint destruction of chronic synovitis.     

The reactions of 1,10-phenanthroline with yeast alcohol dehydrogenase.

Freshly prepared samples of yeast alcohol dehydrogenase (EC 1.1.1.1) were inhibited by 1,10-phenanthroline at pH 7.0 and 0 degrees C in a two-stage process. The first step appeared to be slowly established, but was rendered reversible by removal of reagent or by addition of excess Zn2+ ions. The second step was irreversible and was associated with the dissociation of the tetrameric enzyme. The presence of saturating concentrations of NAD+ or NADH promoted and enhanced inhibition by the slowly established reversible process, but prevented dissociation of the enzyme. For the incubation mixtures containing NAD+, removal of the 1,10-phenanthroline resulted in virtually complete recovery of activity, whereas, for the incubation mixtures containing NADH, removal of the reagent gave only partial re-activation. The presence of NAD+ and pyrazole, or NADH and acetamide, in incubation mixtures with the enzyme gave rise to ternary complexes that gave protection against both forms of inactivation by 1,10-phenanthroline. The results support the view that at least some of the Zn2+ ions associated with yeast alcohol dehydrogenase have a catalytic, as opposed to a purely structural, role.     

Iron: an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

The activity of the ethylene-forming enzyme (EFE) in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells was almost completely abolished within 10 min by 0.4 mM of the metal-chelating agent 1,10-phenanthroline. Subsequent addition of 0.4 mM FeSO₄ immediately reversed this inhibition. A partial reversion was also obtained with 0.6 mM CuSO₄ and ZnSO₄, probably as a consequence of the release of iron ions from the 1,10-phenanthroline complex. The inhibition was not reversed by Mn²⁺ or Mg²⁺. Tomato cells starved of iron exhibited a very low EFE activity. Addition of Fe²⁺ to these cells caused a rapid recovery of EFE while Cu²⁺, Zn²⁺ and other bivalent cations were ineffective. The recovery of EFE activity in iron-starved cells was insensitive to cycloheximide and therefore does not appear to require synthesis of new protein. The EFE activity in tomato cells was induced by an elicitor derived from yeast extract. Throughout the course of induction, EFE activity was blocked within 10–20 min by 1,10-phenanthroline, and the induced level was equally rapidly restored after addition of iron. We conclude that iron is an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in vivo.