Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.     

The Impact of Biology on Risk Assessment--workshop of the National Research Council''s Board on Radiation Effects Research. July 21-22, 1997, National Academy of Sciences, Washington, DC

Cholinesterases exhibit functions apart from their esterase activity. We have demonstrated an aryl acylamidase and a zinc stimulated metallocarboxypeptidase activity in human serum butyrylcholinesterase. To establish the presence of zinc binding sites in the enzyme we examined the effect of metal chelators on its catalytic activities. The metal chelators 1,10-phenanthroline and N,N,N',N'-tetrakis (2-pyridyl methyl)ethylene diamine (TPEN) inhibited all the three catalytic activities in the enzyme. However, EDTA inhibited the peptidase activity exclusively without affecting the cholinesterase and aryl acylamidase activities. The catalytic activities were recovered upon removal of the chelator by Sephadex G-25 chromatography. Pre-treatment of the enzyme with any one of the three chelators resulted in the binding of the enzyme to a zinc-Sepharose column or to 65Zn2+. Histidine modification of the enzyme pretreated with chelators resulted in abolition of 65Zn2+ binding and zinc-Sepharose binding. Whereas the binding studies demonstrated removal of a metal from a Zn2+ binding site, attempts to remove the metal responsible for catalytic activity were unsuccessful. Atomic absorption spectroscopy indicated approximately 2.5 mol of zinc per mol of enzyme before treatment with EDTA and 1 mol zinc per mol enzyme after EDTA treatment. The results indicate that there are at least two metal binding sites on butyrycholinesterase. The presence of two HXXE...H sequences in butyrylcholinesterase supports these findings. Our studies implicate a zinc dependent metallocarboxypeptidase activity in the non-cholinergic functions of butyrylcholinesterase.     

Iodine-123 labelled nor-beta-CIT binds to the serotonin transporter in vivo as assessed by biodistribution studies in rats

Flavobacterium aurantiacum NRRL B-184 possesses the ability to degrade aflatoxin B1 in solution and in several food items. Aflatoxin B1 is a potent carcinogen that causes significant economic losses to the agricultural and food industry. The role of trace metal ions (Cu2+, Mn2+, Zn2+, and Co2+) were studied in an effort to understand the enzymatic system involved in aflatoxin B1 degradation by F aurantiacum. The effect of divalent chelators (EDTA and 1,10-phenanthroline [OPT]) in the presence of the trace metal ions was studied as well. Aflatoxin B1 (10 microg/ml) was added to 72-h cultures of F aurantiacum that had been washed and resuspended in phosphate buffer (pH 7.0). HPLC was used to determine aflatoxin B1 concentration in these cultures. Incubating cells at 30 degrees C with 1 and 10 mM Cu2+, Mn2+, and Zn2+ significantly decreased aflatoxin B degradation after 4 and 24 h (P < 0.05). Decreased degradation was also observed with 1 and 10 mM Cu2+ and Zn2+ after 48 h and with 0.1 mM Cu2+ after 24 and 48 h. Co2+ did not have a significant effect on aflatoxin B1 degradation. EDTA and OPT did not counter the inhibition in the presence of Cu2+. The addition of 1 mM EDTA countered the inhibition by 1 mM Mn2+ after 4 and 24 h, but 1 mM OPT did not counter the inhibition by 10 mM Mn2+ after 4 and 24 h. OPT countered the inhibition by 1 mM Zn2+ after 4 and 48 h. These trace elements inhibit aflatoxin B1 degradation by F aurantiacum. In addition, their presence necessitates higher concentrations (>1 mM) of EDTA and OPT for the removal of their inhibitory effect.     

Productive and persistent infection of human thymic epithelial cells in vitro with HIV-1

Clostridium sporogenes was isolated from rabbits with antibiotic-associated hemorrhagic diarrhea, then its hemorrhagic toxin was purified from the supernatant by means of hydrophobic interaction chromatography, hydroxyapatite chromatography, and gel filtration. The purified toxin's hemorrhagic activity was completely inhibited by EDTA, but not by PMSF or ovomucoid; and fully restored by the addition of such divalent cations as Zn2+, Ca2+ and Mg2+. The purified toxin did not hydrolyze azocasein, or type I or II collagens. But the purified toxin did hydrolyze type III and IV collagens, and also gelatins prepared from type I, II, III and IV collagens. It appears, therefore that C. sporogenes's toxin is a collagenase that hydrolyzes type III and IV collagens, which are major constituents of the tunica intima and media of blood vessels. And that fact suggests that hemorrhage caused by the toxin depend on its collagenase activity.     

An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex

Calcyclin (CaCY) is a member of the S100 subfamily of helix-loop-helix (EF-hand) calcium-binding proteins. Human CaCY was overexpressed in Escherichia coli and purified with an overall yield of 40 mg/l culture. Ca2+ and Zn2+ binding properties of CaCY were examined with respect to the oxidation state of the single Cys residue at position 3. CaCY with the SH group either reduced, blocked or oxidized stays as a dimer as shown by analytical ultracentrifugation. Upon binding of Ca2+, CaCY exhibits 30% enhancement of the Tyr fluorescence, the apparent binding constant (Ka) being 2.8-5.8x10(4) M(-1). Oxidized CaCY binds Ca2+ approximately twice as weakly than its reduced form. The affinity for Ca2+ is increased in the presence of caldesmon, which could be a potential target molecule. Fully reduced CaCY binds Zn2+ with an affinity of at least 1.0x10(7) M(-1). As compared to Ca2+, Zn2+ binding results in a three times greater enhancement of the Tyr fluorescence. Saturation occurs at a Zn2+/CaCY ratio of 2:1. The reactivity of Cys3 is reduced by Zn2+ binding, although oxidized CaCY still binds Zn2+. On the basis of the effects of thiol-directed labels on the affinities for Ca2+ and Zn2+, the fluorescence changes accompanying the binding, and the CaCY reactivity with a hydrophobic probe, it was concluded that the two cations bind to CaCY at different sites: Ca2+ binds probably at the EF-hand type sites, whereas binding of at least one Zn2+ ion involves the Cys residue, and results in a different structural change.     

Functional consequence of mutating conserved residues of the yeast farnesyl-protein transferase beta-subunit Ram1(Dpr1)

Ras proteins, fungal mating pheromones, and other proteins terminating in the sequence CaaX (where C is Cys, a is any aliphatic amino acid, and X is the C-terminal residue) are posttranslationally prenylated. Farnesyl-protein transferase (FPTase) transfers the farnesyl moiety of farnesyl pyrophosphate (FPP) to the thiol of the CaaX box cysteine in a reaction that requires Zn2+ and Mg2+. We have created mutations in conserved amino acids of the yeast Ram1 protein to identify residues important for Zn2+-dependent FPTase activity. Wild-type and mutant Ram1 proteins were expressed as operon fusions in bacteria, and FPTase activity was measured. Mutations in conserved residues Glu256, His258, Asp307, Cys309, Asp360, and His363 reduce FPTase activity. Asp307, Cys309, and His363 correspond to the residues that have been shown to coordinate Zn2+ in mammalian FPTase. The H258N mutant enzyme exhibited an increased sensitivity to the Zn2+ chelator 1,10-phenanthroline, required higher concentrations of Zn2+ to restore activity to the apoenzyme, and had a 10-fold reduction in catalytic efficiency. The decreases in FPTase activity observed do not appear to be caused by major structural perturbations because the mutants were stably expressed and retained the ability to interact with Ram2p during purification. The FPTase activity of the mutants measured in vitro correlated well with their ability to complement the mating and growth defects of a ram1Delta strain in vivo.     

A 3D deformable surface model for segmentation of objects from volumetric data in medical images

Type A Clostridium botulinum, the causative agent of the food poisoning botulism disease, secretes botulinum neurotoxins along with seven neurotoxin associated proteins (NAPs). The function of NAPs has been shown to protect the neurotoxin from acidity, heat, and proteolytic attack in the environmental and gastrointestinal tract during the toxicogenesis of the botulism disease. One of the NAPs, purified from type A botulinum neurotoxin complex, showed hemagglutination activity. A direct interaction has been demonstrated between purified NAP, a 33-kDa hemagglutinin or Hn-33, and the neurotoxin by using Sephadex G-200 column chromatography. Furthermore, Hn-33 has complete resistance against proteolytic attack at pH 2.0 as well as at normal physiological pH. We have investigated digestion of the neurotoxin in the presence and absence of Hn-33. The neurotoxin alone has been found to be more susceptible to the enzymatic digestion than neurotoxin with Hn-33. The presence of Hn-33 changes the proteolytic fragmentation pattern of the neurotoxin. It seems that Hn-33 protects the neurotoxin from proteolysis either by structural modification of the neurotoxin or by blocking the protease accessible sites of the neurotoxin.     

Impaired cerebellar long-term potentiation in type I adenylyl cyclase mutant mice

In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5mM EGTA, only the parental strain was able to take up 65Zn2+. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI) and localized at 42 min on the genetic map of E. coli. At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 microM Zn2+. A znuA-lacZ operon fusion was repressed by 5 microM Zn2+ and showed a more than 20-fold increase in beta-galactosidase activity when Zn2+ was bound to 1.5 microM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB.     

The Azotobacter vinelandii mannuronan C-5-epimerase AlgE1 consists of two separate catalytic domains

The Azotobacter vinelandii enzyme AlgE1 is a member of a family of secreted mannuronan C-5-epimerases. These enzymes convert beta-D-mannuronic acid residues (M) to alpha-L-guluronic acid residues (G) at the polymer level in the industrially important polysaccharide alginate, leading to altered physical and immunological properties of the polymer. The reaction product of AlgE1 was found to be a mixture of blocks of continuous G residues (G-blocks) and blocks containing alternating M and G residues (MG-blocks). The enzyme is dependent on Ca2+ for activity, and only Sr2+ of those tested was able to replace Ca2+. Zn2+ blocked the activity even at low concentrations. algE1 has been divided into two parts based on the modular type of structure previously reported to be a characteristic of the secreted epimerases, and each part has been expressed in Escherichia coli. These experiments showed that AlgE1 contains two catalytic domains, AlgE1-1, which introduces both G-blocks and MG-blocks, and AlgE1-2, which only introduces MG-blocks. AlgE1-1 has a much lower specific activity than both AlgE1-2 and AlgE1. However, the two halves of AlgE1 seem to cooperate in such a way that they contribute approximately equally to the overall epimerization reaction.     

Change in conformation of plasma membrane phospholipid scramblase induced by occupancy of its Ca2+ binding site

Phospholipid (PL) scramblase is a 35.1 kDa plasma membrane protein that mediates the accelerated transbilayer migration of plasma membrane PL in activated, injured, or apoptotic cells exposed to elevated intracellular Ca2+. We recently identified a conserved segment in the PL scramblase polypeptide (residues Asp273 to Asp284) that is essential for its PL-mobilizing function and was presumed to contain the Ca2+ binding site of the protein (Zhou, Q., Sims, P. J., and Wiedmer, T. (1998) Biochemistry 37, 2356-2360). Whereas the sequence of this peptide segment resembles that of known Ca2+-binding loops within EF-hand containing proteins, it is unusual in being a single such loop in the entire protein and in being closely spaced to the predicted transmembrane helix (Ala291-Gly309). To gain insight into how Ca2+ activates the PL-mobilizing function of PL scramblase, we analyzed conformational changes associated with occupancy of this putative Ca2+ binding site. In addition to activation by Ca2+, the PL-mobilizing function of PL scramblase was found to be activated by other ions, with apparent affinities Tb3+, La3+ > Ca2+ > Mn2+ > Zn2+ > Sr2+ > Ba2+, Mg2+. Evidence for coordinate binding of metal ion by the polypeptide was provided by resonance energy transfer from protein Trp to Tb3+, which was competed by excess Ca2+. Metal binding to PL scramblase was accompanied by increased right-angle light scattering and by a prominent change in circular dichroism, suggesting that coordinate binding of the metal ion induces a conformational change that includes self-aggregation of the polypeptide. Consistent with this interpretation, addition of Ca2+ was found to protect PL scramblase from proteolysis by trypsin both in detergent solution as well as in situ, within the erythrocyte membrane. Mutation in the segment Asp273-Asp284 reduced Tb3+ incorporation and attenuated the change in CD spectrum induced by bound metal ligand, confirming that this suspected EF-hand loopike segment of the polypeptide directly contributes to the Ca2+ binding site.     

Mechanism of Ca2+ and monosaccharide binding to a C-type carbohydrate-recognition domain of the macrophage mannose receptor

Site-directed mutagenesis has been used to identify residues that ligate Ca2+ and sugar to the fourth C-type carbohydrate-recognition domain (CRD) of the macrophage mannose receptor. CRD-4 is the only one of the eight CRDs of the mannose receptor to exhibit detectable monosaccharide binding when expressed in isolation, and it is central to ligand binding by the receptor. CRD-4 requires two Ca2+ for sugar binding, like the CRD of rat serum mannose-binding protein (MBP-A). Sequence comparisons between the two CRDs suggest that the binding site for one Ca2+, which ligates directly to the bound sugar in MBP-A, is conserved in CRD-4 but that the auxiliary Ca2+ binding site is not. Mutation of the four residues at positions in CRD-4 equivalent to the auxiliary Ca2+ binding site in MBP-A indicates that only one, Asn728, is involved in ligation of Ca2+. Alanine-scanning mutagenesis was used to identify two other asparagine residues and one glutamic acid residue that are probably involved in ligation of the auxiliary Ca2+ to CRD-4. Sequence comparisons with other C-type CRDs suggest that the proposed binding site for the auxiliary Ca2+ in CRD-4 of the mannose receptor is unique. Evidence that the conserved Ca2+ in CRD-4 bridges between the protein and bound sugar in a manner analogous to MBP-A was obtained by mutation of one of the amino acid side chains at this site. Ring current shifts seen in the 1H NMR spectra of methyl glycosides of mannose, GlcNAc, and fucose in the presence of CRD-4 and site-directed mutagenesis indicate that a stacking interaction with Tyr729 is also involved in binding of sugars to CRD-4. This interaction contributes about 25% of the total free energy of binding to mannose. C-5 and C-6 of mannose interact with Tyr729, whereas C-2 of GlcNAc is closest to this residue, indicating that these two sugars bind to CRD-4 in opposite orientations. Sequence comparisons with other mannose/GlcNAc-specific C-type CRDs suggest that use of a stacking interaction in the binding of these sugars is probably unique to CRD-4 of the mannose receptor.     

Phosphorylation of 25-kDa synaptosome-associated protein. Possible involvement in protein kinase C-mediated regulation of neurotransmitter release

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells. Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced [3H]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25. A phosphorylation is likely to occur at Ser187, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins. SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation. These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.     

Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis

The cortical deposition of Abeta is an event that occurs in Alzheimer's disease, Down's syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Abeta, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Abeta at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Abeta1-40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Abeta1-40 bound three to four Cu2+ ions when precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low nanomolar concentrations of both Abeta1-40 and Abeta1-42 with submicromolar concentrations of Cu2+. Unlike Abeta1-40, Abeta1-42 was precipitated by submicromolar Cu2+ concentrations at pH 7.4. Rat Abeta1-40 and histidine-modified human Abeta1-40 were not aggregated by Zn2+, Cu2+, or Fe3+, indicating that histidine residues are essential for metal-mediated Abeta assembly. These results indicate that H+-induced conformational changes unmask a metal-binding site on Abeta that mediates reversible assembly of the peptide. Since a mildly acidic environment together with increased Zn2+ and Cu2+ are common features of inflammation, we propose that Abeta aggregation by these factors may be a response to local injury. Cu2+, Zn2+, and Fe3+ association with Abeta explains the recently reported enrichment of these metal ions in amyloid plaques in Alzheimer's disease.     

SNAP-25 is required for a late postdocking step in Ca2+-dependent exocytosis

The Ca2+-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca2+-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile181-Gln197) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca2+-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.     

Production of monoclonal antibodies specific to Clostridium botulinum type B neurotoxin

Four monoclonal antibodies were produced for use in a rapid method to detect Clostridium botulinum type B neurotoxin. Cells of mouse myeloma cell line SP2/0 were fused with splenocytes of immunized BALB/c mice. An immunoblot assay of semipurified commercial neurotoxins of C. botulinum types A, B, C, D, E, and F was used to show specificity. All the monoclonal antibodies reacted with type B neurotoxin but did not cross-react with the other types. The monoclonal antibodies, separately and combined, did not neutralize the toxin in mice, and all showed specificity to the whole neurotoxin molecule and the heavy-chain component by immunoblot. No evidence of specific binding to the hemagglutinin molecule was noted. When tested against concentrated cultured supernatants of C. botulinum types A, B, E, and F, the 4 monoclonal antibodies reacted only against type B strains. They will be incorporated into a rapid assay with other specific monoclonal antibodies to detect C. botulinum neurotoxins from pure cultures or suspect foods.     

The CorA Mg2+ transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the third membrane domain identifies a Mg2+ pore

The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10-50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+ and Co2+ was decreased 3-10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2-5-fold. Projection of the third transmembrane segment as an alpha-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the alpha-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA.     

A carboxyl-terminal Cys2/His2-type zinc-finger motif in DNA primase influences DNA content in Synechococcus PCC 7942

The DNA primase gene, dnaG, has been isolated from the cyanobacterium Synechococcus PCC 7942. It is not part of a macromolecular synthesis operon but is co-transcribed with pheT and located adjacent to the metallothionein divergon, smt. At the carboxyl terminus of this DnaG is a Cys2/His2 zinc-finger motif. The carboxyl-terminal 91 residues bound 65Zn and 0.95 g atom of Zn2+ mol-1 were detected with 4-(2-pyridylazo)resorcinol. Following exposure to Cd2+, 0.95 g atom of Cd2+ was displaced by 2 equivalents of p-(hydroxymercuri) phenylsulfonate mol-1, while only 0.03 g atom of Cd2+ was displaced mol-1 polypeptide missing the carboxyl-terminal (residue 592 onward) zinc-finger motif. Zn2+ caused an increase in intensity, and a reduction in wavelength, of Trp fluorescence at the tip of the predicted zinc-finger, while EDTA caused the converse. Cells containing a single chromosomal codon substitution (C597S), altering the zinc-finger, were generated by exploiting Zn2+-sensitive smt mutants and the proximity of dnaG to smt. Cells in which smt and dnaG(C597S) had integrated into the chromosome were selected via restored Zn2+ tolerance. Synechococcus PCC 7942 and its dnaG(C597S) mutant grew at equivalent rates, but the latter had a reduced number of chromosomes.     

Two cell-wall-associated aminopeptidases from Lactobacillus helveticus and the purification and characterization of APII from strain ITGL1

Lactobacillus helveticus ITGL1 is able to hydrolyse many amino-acyl and dipeptidyl-p-nitroanilides. Analysis of heat inactivation kinetics, metal ion and protease inhibitor effects, and the subcellular location of aminopeptidase activities in both the parental strain and mutants deficient in lysyl-p-nitroanilide hydrolysis, led to the characterization of two cell-wall-associated aminopeptidases, APII and APIV. APII, which catalysed L-lysine p-nitroanilide hydrolysis, was purified about 28-fold to homogeneity from cell-wall extracts of L. helveticus ITGL1 and characterized. The purified enzyme appeared to be monomeric, with a molecular mass of 97 kDa. Aminopeptidase activity was greatest at pH 6.5 and 50 degrees C. APII was completely inhibited by bestatin, chelating agents such as EDTA or 1,10-phenanthroline and the divalent cations Zn2+ and Cu2+. The activity of the EDTA-treated enzyme was restored by Co2+, Ca2+ or Mn2+. Although APII was able to degrade several dipeptides and tripeptides with hydrophobic N-terminal amino acid (Leu, Ala), it was inactive on peptides containing Pro or Gly, and may thus contribute to the development of cheese flavour by processing bitter peptides.     

Zinc stimulates Mg2+-dependent 3'-processing activity of human immunodeficiency virus type 1 integrase in vitro

Human immunodeficiency virus type 1 integrase (HIV-1 IN) catalyzes both 3'-donor processing and strand transfer reactions. Previous studies have determined that the N-terminal region, a putative zinc finger, is capable of binding Zn2+. The function of zinc coordination to this domain, however, is still unknown. In this report, we present evidence that Mg2+-dependent 3'-donor processing by HIV-1 IN is enhanced by the addition of Zn2+ in vitro. This activity is inhibited in the presence of the chelator 1,10-phenanthroline (OP). In addition, the Mg2+-dependent 3'-donor processing activity is more sensitive to the concentration of IN than is the Mn2+-dependent activity. A combination of dimethyl sulfoxide (DMSO) and poly(ethylene glycol) (PEG) was found to further activate the Mg2+-dependent 3'-donor processing activity while diminishing the Mn2+-dependent activity. These results suggest factors such as substrate-length, concentration of IN, Zn2+ coordination, and protein-protein interactions are important for efficient and specific donor processing activity with Mg2+ in vitro.