Micronucleated erythrocyte frequency in control and azidothymidine-treated Tk+/+, Tk+/- and Tk-/- mice

The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5′-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk^+/+, Tk^+/− and Tk^−/− mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk^+/+ and Tk^+/− mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk^−/− mice. Unexpectedly, neonatal Tk^−/− mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk^+/+ and Tk^+/− mice (e.g., 3.4% MN-RETs and 4.8% MN-NCEs in Tk^−/− mice versus 0.7 and 0.6% MN-RETs and MN-NCEs in neonatal Tk^+/+ mice). Additional assays performed on untreated Tk^−/− mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk^−/− mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk^−/− mice suggest the presence of an endogenous genotoxic activity in these mice.     

Effect of p53 genotype on gene expression profiles in murine liver

The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the “guardian of the genome”. Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53^−/− and p53^+/− mice. Six male mice from each genotype (p53^+/+, p53^+/−, and p53^−/−) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53^+/+ and p53^+/− or between p53^+/+ and p53^−/− at the level of p ≤ 0.05. Both genes with increased expression and decreased expression were identified in p53^+/− and in p53^−/− mice. Most notable in the gene list derived from the p53^+/− mice was the significant reduction in p53 mRNA. In the p53^−/− mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.     

The basal levels of 8-oxoG and other oxidative modifications in intact mitochondrial DNA are low even in repair-deficient (Ogg1(-/-)/Csb(-/-)) mice

Mitochondrial DNA (mtDNA) is assumed to be highly prone to damage by reactive oxygen species (ROS) because of its location in close proximity to the mitochondrial electron transport chain. Accordingly, mitochondrial oxidative DNA damage has been hypothesized to be responsible for various neurological diseases, ageing and cancer. Since 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most frequent oxidative base modifications, is removed from the mitochondrial genome by the glycosylase OGG1, the basal levels of this lesion are expected to be highly elevated in Ogg1^−/− mice. To investigate this hypothesis, we have used a mtDNA relaxation assay in combination with various repair enzymes (Fpg, MutY, endonuclease III, endonuclease IV) to determine the average steady-state number of oxidative DNA modifications within intact (supercoiled) mtDNA from the livers of wild-type mice and those deficient in OGG1 and/or the Cockayne syndrome B (CSB) protein for mice aged up to 23 months. The levels of all types of oxidative modifications were found to be less than 12 per million base pairs, and the difference between wild-type and repair-deficient (Ogg1^−/−/Csb^−/−) mice was not significant. Thus, the increase of 8-oxoG caused by the repair deficiency in intact mtDNA is not much higher than in the nuclear DNA, i.e., not more than a few modifications per million base pairs. Based on these data, it is hypothesized that the load of oxidative base modifications in mtDNA is efficiently reduced during replication even in the absence of excision repair.     

Spontaneous hypomorphic mutations in antioxidant enzymes of mice

An antioxidant enzymatic system is pivotal for aerobic animals to minimize the damage induced by reactive oxygen species. Spontaneous mutant animals with altered antioxidant enzyme activity should be useful for the study of the function of these enzymes in vivo. We examined the nucleotide sequences of the genes for the major antioxidant enzymes, including catalase (Cat), superoxide dismutase (Sod1, Sod2, Sod3), glutathione peroxidase (Gpx1, Gpx2, Gpx3, Gpx4, Gpx5), and glutathione reductase (Gsr) in 10 inbred mouse strains. Nonsynonymous nucleotide polymorphisms were identified in all genes, except for Gpx1, Gpx3, and Gpx4. Notably, the SJL/J mouse strain possessed unique nucleotide substitutions in the Gsr and Sod2 genes, which led to Asp39Ala and Val138Met amino acid substitutions in GSR and SOD2, respectively. The specific activity of GSR of SJL/J mice was reduced to 65% of that of NZB/N mice. In vivo activity, however, was higher in SJL/J, due to upregulated expression of the enzyme. The SOD2 activity in SJL/J mice was reduced to half that of other mouse strains. Consistent with this reduction, oxidative damage in the mitochondria was increased as demonstrated by a decrease of total glutathione and an increase in the levels of protein oxidation. These spontaneous hypomorphic alleles would be valuable in the study of free radical biology.     

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt^+/−→aprt^−/−) was 1.3 × 10⁻⁵/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt^−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.     

Rate constants and activation parameters for organo-cobalt porphyrin bond homolysis from NMR relaxation times

¹H NMR line broadening is found to be an effective complimentary method to chemical trapping for determining the rates and activation parameters for organo-metal bond homolysis events that produce freely diffusing radicals. Application of this method is illustrated by measurement of bond homolysis activation parameters for a series of organo-cobalt porphyrin complexes ((TPP)Co-C(CH₃)₂CN (ΔH^≠ = 19.5±0.9 kcal mol⁻¹, ΔS^≠ = 12±3 cal°K⁻¹ mol⁻¹), (TMP)Co-C(CH₃)₂CN (ΔH^≠ = 20±1 kcal mol⁻¹,ΔS^≠ = 13±2 cal°K⁻¹ mol⁻¹), (TAP)Co-C(CH₃)₂CO₂CH₃ (ΔH^≠ = 18.2±0.5 kcal mol⁻¹, ΔS^≠ = 12±2 cal °K⁻¹ mol⁻¹), (TAP)Co-CH(CH₃)C₆H₅ (ΔH^≠ = 22.5±0.5, ΔS^≠ = 17±2 cal °K⁻¹ mol⁻¹)). The line broadening method is particularly useful in determining activation parameters for dissociation of weakly bonded organometallics where the rate of homolysis can exceed the range measurable by conventional chemical trapping methods.     

Pressure effects on the rates of Hg-assisted chloride release from some [CrCl(diamine)(triamine)] complexes

The rate of Hg²⁺-assisted chloride release from several mer-[CrCl(diamine)(triamine)]²⁺ complexes has been measured as a function of pressure, Hg²⁺ concentration and temperature. The calculated activation volumes are independent of [Hg²⁺] and temperature and kinetic parametes 10⁴ k_Hg (25 °c) (M⁻¹ s⁻¹), ΔH^‡ (kJ mol⁻¹), ΔS^‡ (J K⁻¹ mol⁻¹), ΔV^‡ (cc mol⁻¹) are: (en)(dpt): 6.44. 75.5, −52, −5.0; (ibn)(dpt): 5.81, 89.5, −6, −0.03; (Me₂tn)(dpt): 22.2, 84.9, −11, −0.5; (tn)(dpt): 29.1, 87, −1, +0.3; (en)(2,3-tri): 1.94, 87.0, −24, −5.7; (en)(Medpt): 0.417, 94.6, −11, −0.8; (tn)(Medpt): 9.14, 98.3, +26, +1.8.     

Proton nuclear magnetic resonance study of N,N-diethylformamide exchange on (N,N-diethylformamide)2,2′2″-tris(dimethylamino)triethylamine)cobalt(II) and its copper(II) analogue

Proton NMR studies of N,N-diethylformamide (def) exchange on [M(Me₆tren)def]²⁺ where M = Co and Cu yield: k_ex (298.2K) = 26.3 ± 2.2, 980 ± 70 s⁻¹; ΔH^≠ = 58.3 ± 1.7, 36.3 ± 0.9 kJ mol⁻¹; ΔS^≠= −22.2 ± 4.6, −65.9 ± 2.5 J K⁻¹ mol⁻¹; and ΔV^≠ = −1.3 ± 0.2, 5.3 ± 0.3 cm³ mol⁻¹ respectively. These data which are consistent with a and d activation modes operating when M = Co and Cu respectively are compared with data for related systems.     

Increased energy expenditure, dietary fat wasting, and resistance to diet-induced obesity in mice lacking renin

An overactive renin-angiotensin system is associated with obesity and the metabolic syndrome. However, the mechanisms behind it are unclear. Cleaving angiotensinogen to angiotensin I by renin is a rate-limiting step of angiotensin II production, but renin is suggested to have angiotensin-independent effects. We generated mice lacking renin (Ren1c) using embryonic stem cells from C57BL/6 mice, a strain prone to diet-induced obesity. Ren1c^−/− mice are lean, insulin sensitive, and resistant to diet-induced obesity without changes in food intake and physical activity. The lean phenotype is likely due to a higher metabolic rate and gastrointestinal loss of dietary fat. Most of the metabolic changes in Ren1c^−/− mice were reversed by angiotensin II administration. These results support a role for angiotensin II in the pathogenesis of diet-induced obesity and insulin resistance.     

3nion-independent conformational ordering in iota-carrageenan: Disorder-order equilibria and dynamics

The equilibria and dynamics of the disorder-to-order transition of the anionic polysaccharide iota-carrageenan have been studied in the presence of tetramethyl-ammonium salts. By the use of a stopped-flow polarimeter, the rate equation and temperature dependence of the observed forward rate-constant were found to accord with a co-operative dimerisation process. Activation parameters for helix nucleation were shown to be independent of the anion for solutions containing tetramethylammonium chloride and bromide, i.e., ΔH^‡ = 1 ±3 kJ.mol⁻¹, ΔS^‡ = −178 ±10 J.mol⁻¹.K⁻¹, ΔG^‡_298K = 54 ±2 kJ.mol⁻¹, and k_nuc,298K = 1880 ±80 dm³.mol⁻¹.s⁻¹. The temperature dependence of optical rotation was also shown to be independent of the anion present.     

Nickel(II) and copper(II) complexes of mixed benzene-triazolehemiporphyrazines

The kinetics of substitution reactions of [η-CpFe(CO)₃]PF₆ with PPh₃ in the presence of R-PyOs have been studied. For all the R-PyOs (R = 4-OMe, 4-Me, 3,4-(CH)₄, 4-Ph, 3-Me, 2,3-(CH)₄, 2,6-Me₂, 2-Me), the reactions yeild the same product [η⁵-CpFe(CO)₂PPh₃]PF₆, according to a second-order rate law that is first order in concentrations of [η⁵-CpFe(CO)₃]PF₆ and of R-PyO but zero order in PPh₃ concentration. These results, along with the dependence of the reaction rate on the nature of R-PyO, are consistent with an associative mechanism. Activation parameters further support the bimmolecular nature of the reactions: ΔH^≠ = 13.4 ± 0.4 kcal mol⁻¹, ΔS^≠ = −19.1 ± 1.3 cal k⁻¹ mol⁻¹ for 4-PhPyO; ΔH^≠ = 12.3 ± 0.3 kcal mol⁻¹, ΔS^≠ = 24.7 ±1.0 cal K⁻¹ mol⁻¹ for 2-MePyO. For the various substituted pyridine N-oxides studied in this paper, the rates of reaction increase with the increasing electron-donating abilities of the substituents on the pyridine ring or N-oxide basicities, but decrease with increasing ¹⁷O chemical shifts of the N-oxides. Electronic and steric factors contributing to the reactivity of pyridine N-oxides have been quantitatively assessed.     

Kinetics and mechanism of the stoichiometric oxygenation of [Cu(fla)(idpa)]ClO4 [fla=flavonolate, IDPA=3,3′-imino-bis(N,N-dimethylpropylamine)] and the [Cu(fla)(idpa)]ClO4-catalysed oxygenation of flavonol

Oxygenation of [Cu^II(fla)(idpa)]ClO₄ (fla=flavonolate; IDPA=3,3′-iminobis(N,N-dimethylpropylamine)) in dimethylformamide gives [Cu^II(idpa)(O-bs)]ClO₄ (O-bs=O-benzoylsalicylate) and CO. The oxygenolysis of [Cu^II(fla)(idpa)]ClO₄ in DMF was followed by electronic spectroscopy and the rate law −d[{Cu^II(fla)(idpa)}ClO₄]/dt=k_obs[{Cu^II(fla)(idpa)}ClO₄][O₂] was obtained. The rate constant, activation enthalpy and entropy at 373 K are k_obs=6.13±0.16×10⁻³ M⁻¹ s⁻¹, ΔH^‡=64±5 kJ mol⁻¹, ΔS^‡=−120±13 J mol⁻¹ K⁻¹, respectively. The reaction fits a Hammett linear free energy relationship and a higher electron density on copper gives faster oxygenation rates. The complex [Cu^II(fla)(idpa)]ClO₄ has also been found to be a selective catalyst for the oxygenation of flavonol to the corresponding O-benzoylsalicylic acid and CO. The kinetics of the oxygenolysis in DMF was followed by electronic spectroscopy and the following rate law was obtained: −d[flaH]/dt=k_obs[{Cu^II(fla)(idpa)}ClO₄][O₂]. The rate constant, activation enthalpy and entropy at 403 K are k_obs=4.22±0.15×10⁻² M⁻¹ s⁻¹, ΔH^‡=71±6 kJ mol⁻¹, ΔS^‡=−97±15 J mol⁻¹ K⁻¹, respectively.     

Studies on the kinetics and mechanism of anation reactions of some six-coordinate complexes of chromium(III) in aqueous solution

Rates of stepwise anation of cis-Cr(ox)₂(H₂O₂)⁻ with SCN⁻/N₃⁻, Cr(acac)₂(H₂O)₂⁺ with SCN⁻ and Cr(atda)(H₂O)₂ with SCN⁻ have been investigated in weakly acidic aqueous solutions. Rate constants, k_I and k_II for the two steps in each system, are composite as k_x = k_x⁰+k_x^X[X⁻] (x = I, II; X⁻ = SCN⁻, N₃⁻). These rate constants have been evaluated also as the corresponding ΔH^≠ and ΔS^≠ values. The results obtained and the plausible I_d mechanism seem to suggest Cr---OOC bond dissociation (hence a strongly negative ΔS^≠) generating the transition state in each system with outer-sphere association forming the precursor complex in the X⁻ dependent paths.     

Model reactions of a carbonylation catalyst: phosphite induced migratory CO insertion in [MeIr(CO)2I3]

Carbonylation of the anionic iridium(III) methyl complex, [MeIr(CO)₂I₃]⁻ (1) is an important step in the new iridium-based process for acetic acid manufacture. A model study of the migratory insertion reactions of 1 with P-donor ligands is reported. Complex 1 reacts with phosphites to give neutral acetyl complexes, [Ir(COMe)(CO)I₂L₂] (L = P(OPh)₃ (2), P(OMe)₃ (3)). Complex 2 has been isolated and fully characterised from the reaction of Ph₄As[MeIr(CO)₂I₃] with AgBF₄ and P(OPh)₃; comparison of spectroscopic properties suggests an analogous formulation for 3. IR and ³¹P NMR spectroscopy indicate initial formation of unstable isomers of 2 which isomerise to the thermodynamic product with trans phosphite ligands. Kinetic measurements for the reactions of 1 with phosphites in CH₂Cl₂ show first order dependence on [1], only when the reactions are carried out in the presence of excess iodide. The rates exhibit a saturation dependence on [L] and are inhibited by iodide. The reactions are accelerated by addition of alcohols (e.g. 18× enhancement for L = P (OMe)₃ in 1:3 MeOH-CH₂Cl₂). A reaction mechanism is proposed which involves substitution of an iodide ligand by phosphite, prior to migratory CO insertion. The observed rate constants fit well to a rate law derived from this mechanism. Analysis of the kinetic data shows that k₁, the rate constant for iodide dissociation, is independent of L, but is increased by a factor of 18 on adding 25% MeOH to CH₂Cl₂. Activation parameters for the k₁ step are ΔH^≠ = 71 (±3) kJ mol⁻, ΔS^≠ = −81 (±9) J mol⁻¹ K⁻¹ in CH₂Cl₂ and ΔH^≠ = 60(±4) kJ mol⁻¹, ΔS^≠ = −93(± 12) J mol⁻¹ K⁻¹ in 1:3 MeOH-CH₂Cl₂. Solvent assistance of the iodide dissociation step gives the observed rate enhancement in protic solvents. The mechanism is similar to that proposed for the carbonylation of 1.     

Structure of 7S seed globulin from Phaseolus vulgaris L. in solution

The structure of the 7S globulin from Phaseoulus vulgaris L in dilatue solutions has been studied by small angle X-ray scattering (SAXS), by quasi-elastic light scattering (Q ELS), by circular dichroism spectroscopy (c.d.), and by precise density measurements. The molar mass, the radius of gyration, the volume, the maximum dimension and the diffusion coefficient were determined as M = 1.45 × 10⁵ g mol⁻¹, R_G = 4.05 nm, V = 300- nm³, L = 13.0 nm and D_20,w⁰ = 4.5 × 10⁻⁷ cm² s⁻¹, respectively. The molecule has an asymmetrical shape with the dimensions 12.5 × 12.5 × 3.75 nm. The secondary structure of the 7S globulin is characterized by a small portion of -helical structure (14%) and a marked content of β-structure (18%).     

Rate of electron transfer between plastocyanin, cytochrome ƒ, related proteins and artificial redox reagents in solution

The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10⁷ M⁻¹ · s⁻¹, at 298 °K and pH 7.0, with activation parameters ΔH^‡ = 44 kJ · mole⁻¹ and ΔS^‡ = +46 J · mole⁻¹ · °K⁻¹. Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10⁵, 7.5 · 10⁵ and 1.0 · 10⁶ M⁻¹ · s⁻¹, respectively.

Similar measurements made with azurin instead of plastocyanin gave k = 6 · 10⁶ and approx. 2 · 10⁷ M⁻¹ · s⁻¹ for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively.

Rate constants of 115 and 80 M⁻¹ · s⁻¹ were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 10⁴ and 8 · 10⁴ M⁻¹ · s⁻¹.

The results are discussed in relation to photosynthetic electron transport.     

Cellular determinants of the mutational specificity of 1-nitroso-6-nitropyrene and 1-nitroso-8-nitropyrene in the lacI gene of Escherichia coli.

We have characterized 202 lacI⁻ mutations, and 158 dominant lacI^−d mutations following treatment of Escherichia coli strains NR6112 and EE125 with 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene. In all, 91% of the induced point mutations occurred at G:C residues. The −(G:C) frameshifts were the dominant mutational class in the lacI⁻ collections of both NR6112 and EE125, and in the lacI^−d collection of NR6112. Frameshift mutations occurred preferentially in runs of guanine residues, and their frequency increased with the length of the reiterated sequence. In strain EE125, which contained the plasmid pKM101, there was a marked stimulation in the frequency of base substitution mutations that was particularly apparent in the lacI^−d collection. This study completes a comprehensive analysis of 1194 lacI⁻ and 348 lacI^−d mutations induced by either 1,6-NONP or its positional isomer 1-nitroso-8-nitropyrene (1,8-NONP) in strains of E. coli that differ with regard to their ability to carry out nucleotide excision repair and/or their ability to express the translesion synthesis DNA polymerase RI (MucAB) encoded by plasmid pKM101. Among the mutations are 763 frameshift mutations, 367 base substitutions and 47 deletions; these mutations have been characterized at more than 300 distinct sites in the lacI gene. Our studies provide detailed insight into the DNA sequence alterations and mutational mechanisms associated with dinitropyrene mutagenesis. We review the mutational spectra, and discuss cellular lesion repair or tolerance mechanisms that modulate the observed mutational specificity.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Metal ion substitution in phospholipase C (Bacillus cereus) and its effect on enzyme stability

The effect of guanidinium chloride solutions on the circular dichroism of native (ZnZn-) and apophospholipase C (Bacillus cereus) indicated marked protein unfolding at denaturant concentrations of 1.4–1.8 M and 0.1–0.6 M, respectively. With the apoenzyme near u.V. region circular dichroism bands remained even after all ordered structure appeared to have been lost. Apophospholipase C bound two equivalents of Ni²⁺, Cd²⁺, Co²⁺, Mn², Pb²⁺ or Cu²⁻, with only the latter metal causing marked changes either in circular dichroism or protein fluorescence relative to the native enzyme. Stability in guanidinium chloride for the metalloforms of phospholipase C decreased in the order: ZnZn->ZnCo->NiNi->CoCo->PbPb->CdCd->MnMn-apoenzyme.