Macrophage spreading in vitro : I. Inducers of spreading

Normal mouse peritoneal macrophages attached to glass were induced to spread by one of the following procedures: treatment with proteolytic enzymes or with dithiothreitol; incubation in media of low ionic strength or at pH around 6.0; or layering of the phagocytes on glass bound antigen-antibody complexes [21–23]. With the exception of spreading induced by proteolytic enzymes, macrophage spreading required the presence of Mg²⁺ while Ca²⁺ was clearly less effective. In all cases spreading was found to be temperature dependent, to be favored by an acid pH of the medium and to be decreased by cytochalasin B or by serum. Three primary mechanisms may underlie induced macrophage spreading: (a) a structural or functional alteration of the macrophage membrane; (b) an effect on the substrate; (c) an intracellular effect. These perturbations could result in increased membrane adhesiveness and/or cell deformability.     

Human monocyte spreading in vitro--inducers and effects on Fc and C3 receptors.

Human peripheral blood monocytes undergo cytoplasmic spreading following attachment to a glass surface. The extent of spreading is greater in the presence of antigen-antibody complexes, manganese, subtilisin and dithiothreitol. The human blood monocyte spreads more rapidly than the mouse peritoneal macrophage and is not inhibited by serum. Fc receptor activity is diminished when spreading is induced by antigen-antibody complexes and is not affected by other inducers. The binding of erythrocytes coated with C3 and the ingestion of latex particles are not inhibited during cytoplasmic spreading.     

Reversibility of phorbol diester-induced macrophage spreading

Phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate induced spreading of mouse macrophages with 50% effective concentrations of 3 nM and 35 nM, respectively. Macrophages treated with 100 or 1000 nM phorbol 12, 13- dibutyrate showed a time related decrease in spreading after washout. Spreading induced by 1, 10, or 100 nM phorbol 12-myristate 13-acetate was irreversible; however, washed phorbol 12,13-dibutyrate-treated cells respread after a second exposure to this compound. Washout of ³[H]phorbol diesters corroborated these observations in that 5% of ³H-phorbol 12-myristate 13-acetate and only 0.1% ³[H]phorbol, 12,13-dibutyrate remained associated with washed cells. Since phorbol 12-myristate 13 acetate is much more lipophilic than phorbol 12,13-dibutyrate, the reversibility of phorbol diester-induced macrophage spreading may depend upon the lipophilicity of the derivative utilized.Abbreviations DMEM Dulbecco's Minimal Essential Medium - PDA phorbol 12,13-diacetate - PDBu phorbol 12, 13-dibutyrate - PMA phorbol 12 myristate, 13 acetate - 4PDD phorbol 12, 13 didecanoate     

The Impact of Flap Creation Methods for Sub-Bowman’s Keratomileusis (SBK) on the Central Thickness of Bowman’s Layer

Purpose

To determine the impact of flap creation methods for sub-Bowman’s keratomileusis (SBK) on central Bowman’s layer thickness.

Methods

SBK flaps were made by Moria microkeratome for 20 subjects and by femtosecond (FEMTO) laser for 21 subjects. Corneal sublayer thicknesses were measured by ultra-high resolution optical coherence tomography before SBK and at 1 day, 1 week, 2 weeks, and 1 month afterwards. Each subject was imaged twice on each visit. Thicknesses of central epithelium, Bowman’s layer, flap, and total cornea were calculated using a custom-made automated image processing algorithm. The repeatability of sublayer thickness measurements was tested by the intraclass correlation coefficient (ICC) and by the coefficient of repeatability (CoR) at 1 week post-SBK.

Results

ICCs of the Moria and FEMTO groups were ≥0.959 and ≥0.961 respectively for all sublayer measurements. The segmentation CoRs were less than 6.78% and 5.63% respectively. For both groups, microdistortions were present in the epithelium and Bowman’s layer after SKB. The flap thickness of the Moria group was 9.8 μm (95% confidence interval: 4.8 – 14.8μm) thinner than the FEMTO group one day after SBK (independent samples t-test, P < 0.05). Bowman’s layer became thicker by 1.6 ± 1.1 μm and 1.7 ± 1.6 μm one day post-SBK for the Moria and FEMTO groups (repeated ANOVA, P < 0.05) and then remained stable. Corneal and sublayer thickness were similar between the two groups.

Conclusions

Central Bowman’s layer thickness increased 1 day post-SBK. Flap creation by Moria microkeratome and femtosecond laser did not have significantly different impacts on Bowman’s layer thickness following SBK.

Trial Registration

Chinese Clinical Trial Registry (ChiCTR) NO: ChiCTR-OCH-14004525     

The role of microfilaments in the spreading process of JTC-12 cells

In 5 μg/ml cytochalasin B (CB), spreading of JTC-12 cells over the substratum occurred to some extent, but an almost complete inhibition was seen in 10 μg/ml CB, except for extrusion of thin processes. Formation of microfilament bundles beneath the adhesive surface was correlated with the grade of spreading. Treatment of spreading cells with 10 μg/ml CB caused a retraction of the peripheral cytoplasm or inhibited further spreading and concurrently disintegrated the microfilament bundles. Thus, the circular bundles of the microfilaments inside the cell outline probably enable the concentrical spreading of JTC-12 cells by advancing and consolidating the peripheral cytoplasm.     

Effects of prostaglandins on the spreading, adhesion and migration of mouse peritoneal macrophages

The effects of prostaglandins on the properties of mouse peritoneal macrophages namely spreading, adhesion and migration were investigated. PGE₁ and PGE₂ inhibit the spreading and adhesion of complete Freund's Adjuvant induced peritoneal macrophages significantly at concentrations of 1 ng per ml and above whereas they enhance the migration of these cells at concentrations of 100 ng per ml and above. PGA₂ and PGB₂ are less potent as they inhibit spreading and adhesion only at a concentration of 1 μg per ml. At this concentration PGB₂ enhances migration whereas PGA₂ has no effect. PGF_2α has no effect on the spreading, adhesion and migration of macrophages in the concentration range of 0.1 ng to 1,000 ng per ml.     

Inability of tributyltin-induced chloride/hydroxyl exchange to stimulate calcium transport in mitochondria isolated from flight muscle of the sheep blowfly Lucilia cuprina

Tributyltin in the concentration range 1–4μm failed to stimulate Ca²⁺ transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of P_i, despite its promotion of a rapid Cl⁻/OH⁻ exchange. When 2mm-P_i was present, concentrations of tributyltin greater than 1μm inhibited the initial rate of Ca²⁺ transport and induced efflux of the ion from the mitochondria in Cl⁻- or NO₃⁻-containing media. Lower concentrations had little effect. Oligomycin added at up to 10μg/mg of mitochondrial protein had no effect on Ca²⁺ transport. By contrast, approx. 0.3μm-tributyltin completely inhibited respiration supported by α-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca²⁺ transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl⁻/OH⁻ exchange or producing an oligomycin-like effect.     

Macrophage spreading in vitro. III. The effect of metabolic inhibitors, anesthetics and other drugs on spreading induced by subtilisin

The effect of certain drugs on macrophage spreading induced by the proteolytic enzyme sub-tilisin was quantitatively examined and the 50 and 90 % inhibitory concentrations of the drugs were determined. In most instances the viability of the macrophages was preserved, as shown by phagocytic tests and by experiments in which cells pretreated with the drugs and washed were shown to spread when exposed to subtilisin. Inhibitors of electron transport, oxidative phosphorylation or uncouplers at rather small concentrations all effectively blocked macrophage spreading, indicating an ATP requirement. Spreading was also inhibited by neutral or cationic anesthetics and the reciprocal of their 50 % inhibitory concentrations was linearly related to their octanol-water partition coefficients. Inhibition by the anesthetics paralleled their effects on other membrane phenomena, such as nerve conduction, osmotic lysis of erythrocytes, viral induced cell fusion, or Sarcoma I cell to substrate adhesion, also suggesting a membrane target. Spreading was reduced by the anti-inflammatories indomethacin, or phenylbutazone, by high doses of colchicine or vinblastine, by the putative microfilament-acting drug cytochalasin B or by the SH- reagent n-ethyl maleimide. Colchicine and vinblastine effects may involve mechanisms other than their microtubular actions. Several other drugs, including inhibitors of protein synthesis, did not inhibit the spreading of macrophages. Spreading induced by substrate-bound immune complexes was also inhibited by representative metabolic inhibitors or anesthetics.     

Macrophage arachidonate release via both the cytosolic Ca2+-dependent and -independent phospholipases is necessary for cell spreading

We have observed that phospholipase A₂ (PLA₂) activation and arachidonate (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA₂s in these processes. The PLA₂-specific inhibitors, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL, specific for the Ca²⁺-independent PLA₂ (iPLA₂)) and methyl arachidonoyl fluorophosphonate (MAFP, specific for the Ca²⁺-dependent phospholipase (cPLA₂)) inhibited AA release and cell spreading in a correlated fashion but only modestly decreased cell adherence. Cell spreading was normalized by the addition of AA to PLA₂-inhibited cells. AA release during spreading was also inhibited by Ca²⁺ depletion or protein kinase C (PKC) inhibition, and was accompanied by increased (but transient) phosphorylation of cPLA₂. Inhibition of macrophage spreading, however, only partially inhibited AA release. Moreover, constitutive AA release was seen in fully spread macrophages which was inhibited by BEL, but not MAFP or Ca²⁺ depletion. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the iPLA₂ (which appears to be constitutively active) and cPLA₂ (which appears to be stimulated by adherence/spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA₂.     

Electrophysiology of phagocytic membranes. Role of divalent cations in membrane hyperpolarizations of macrophage polykaryons

The electrophysiological properties of the membrane of mouse peritoneal macrophage polykaryons are studied. Slow hyperpolarizations can be elicited by iontophoretic injections of either Ca2+ or Sr2+ into the cytoplasm. The effect of both cations is identical, since: it is invariably triggered by the cation injection, the amplitude is dependent on the K+ gradient, quinine blocks reversibly the response to both cation injections. Mg2+, Ba2+ and Mn2+ did not elicit responses when injected into the cytoplasm. Ca2+ induced slow hyperpolarizations were reversibly blocked by the addition of Ba2+ to the external saline, but were not affected by the presence of external tetraethylammonium chloride. Cells maintained in saline containing high concentrations of Ca2+, Sr2+ or Mn2+ exhibited sustained hyperpolarizations. Quinine blocked the hyperpolarization induced by high Ca2+ or Sr2+, but was ineffective for the case of Mn2+. Cells hyperpolarized by external Mn2+ frequently exhibited nonlinear, voltage-current characteristics. Similar patterns could also be observed in a small fraction (less than 10%) of the cells in control conditions. Current-induced shifts between two stable membrane potentials were seen either in high Ca2+ or normal medium. The great variability of the responses described for this phagocytic membrane is discussed. The evidence supports the assumption that Ca2+ and Sr2+ can induce transient or persistent hyperpolarized states by activating a potassium permeability. External Mn2+ may act in part by reducing impalement-related current leakage from the phagocytic membrane.     

Use of the green alga Ulva rigida C. Agardh as an indicator species to reassess metal pollution in the Thermaikos Gulf, Greece, after 13 years

The concentrations of metals (Mn, Pb, Fe, Zn, Cu, Cd,Co, Ni, Cr, Na, K, Ca, Mg) were determined in thegreen alga Ulva rigida, in the sediment andseawater of Thermaikos Gulf (Greece) during monthlysamplings in 1994–1995. This Gulf is the recipientof domestic and industrial effluents. Pb, Fe, Cu, Coand Cr concentrations in U. rigida at the studyarea were higher than those 13 years earlier andapparently came from different sources than those forZn, Cd and Ni. The relative abundance of metals inthe alga decreased in the order: Mg > Na > K >Ca > Pb > Fe > Mn > Zn > Cr, Cu > Ni >Co > Cd. Only Cu concentrations in the alga fromKalochori and Cd ones from Viamyl showed significantseasonal changes. Cu and Cd concentrations ingeneral followed the same pattern of variation, withminimum values in winter-spring. This pattern isdiscussed in relation to growth dynamics and tissueage. Only Pb concentrations in the alga showed asignificant positive correlation with concentrationsin the seawater. There were both positive andnegative correlations among some metals in the alga. It is concluded that U. rigida can be used as anindicator species, especially for Pb.     

Chronic exposure of diesel exhaust particles induces alveolar enlargement in mice

Background

Diesel exhaust particles (DEPs) are deposited into the respiratory tract and are thought to be a risk factor for the development of diseases of the respiratory system. In healthy individuals, the timing and mechanisms of respiratory tract injuries caused by chronic exposure to air pollution remain to be clarified.

Methods

We evaluated the effects of chronic exposure to DEP at doses below those found in a typical bus corridor in Sao Paulo (150 μg/m³). Male BALB/c mice were divided into mice receiving a nasal instillation: saline (saline; n = 30) and 30 μg/10 μL of DEP (DEP; n = 30). Nasal instillations were performed five days a week, over a period of 90 days. Bronchoalveolar lavage (BAL) was performed, and the concentrations of interleukin (IL)-4, IL-10, IL-13 and interferon-gamma (INF-γ) were determined by ELISA-immunoassay. Assessment of respiratory mechanics was performed. The gene expression of Muc5ac in lung was evaluated by RT-PCR. The presence of IL-13, MAC2+ macrophages, CD3+, CD4+, CD8+ T cells and CD20+ B cells in tissues was analysed by immunohistochemistry. Bronchial thickness and the collagen/elastic fibers density were evaluated by morphometry. We measured the mean linear intercept (Lm), a measure of alveolar distension, and the mean airspace diameter (D0) and statistical distribution (D2).

Results

DEP decreased IFN-γ levels in BAL (p = 0.03), but did not significantly alter IL-4, IL-10 and IL-13 levels. MAC2+ macrophage, CD4+ T cell and CD20+ B cell numbers were not altered; however, numbers of CD3+ T cells (p ≤ 0.001) and CD8+ T cells (p ≤ 0.001) increased in the parenchyma. Although IL-13 (p = 0.008) expression decreased in the bronchiolar epithelium, Muc5ac gene expression was not altered in the lung of DEP-exposed animals. Although respiratory mechanics, elastic and collagen density were not modified, the mean linear intercept (Lm) was increased in the DEP-exposed animals (p ≤ 0.001), and the index D2 was statistically different (p = 0.038) from the control animals.

Conclusion

Our data suggest that nasal instillation of low doses of DEP over a period of 90 days results in alveolar enlargement in the pulmonary parenchyma of healthy mice.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0172-z) contains supplementary material, which is available to authorized users.     

α-Actinin arcs in megakaryocyte spreading

Cultured megakaryocytes, isolated from guinea pig bone marrow, were treated with buffer or adenosine diphosphate (ADP) (10 μM) on plain or coated glass surfaces. Control cells were rounded and non-adherent. The nucleotide induced the cells to spread to several times the initial diameter, and to become flattened and markedly adherent to the substratum. ‘Cytoskeletons’ were examined by transmission electron microscopy (TEM). Those from unspread cells contained only rare microfilaments and no filament bundles; those from spread cells contained large numbers of microfilaments in nets and many filament bundles, which were largely oriented circumferentially. Interference reflection microscopy demonstrated that the spread cells were attached to the substratum in arc-shaped regions, which corresponded to arcs containing alpha-actinin as seen by specific immunofluorescence of the same cells. However, other arcs of α-actinin staining did not correspond to arcs of tight attachment. We conclude that fibrous arcs, which appear to assemble as part of the spreading process, play a role in what are probably transient surface attachment sites. A survey of observations of spreading in other cell types suggests that similar arcs are more widespread than has been realized.     

Surface spreading as a biotransport mechanism

Surface spreading as a biotransport process is explored. In this model the natural tendency of solutes to spread along interfaces is the driving force for the transport process. Typical spreading velocities were calculated to be of the order of 1 m/s over distances of the order of 10 mm. This represents an effective diffusion coefficient of 10⁴ m²/s — a value which may be two to three orders of magnitude greater than that produced by molecular diffusion. Additional characteristics of surface spreading which are of biological significance are that it is a directed form of transport and it is capable of producing bidirectional flow along a single transport channel. The possibility that surface spreading could be involved as a mechanism in the transport of materials within the main cell body, in axonal transport in nerve cells and in phloem translocation in plants is discussed.     

Metal requirement of the isolated red cell Ca-pump ATPase after elimination of calmodulin dependence by trypsin attack

Mild proteolysis by trypsin activates the purified (Ca²⁺ + Mg²⁺) - ATPase protein from human red cells in a way which is similar to the effect obtained by addition of calmodulin. The trypsin concentration required to reach half maximal effect in 3 minutes at 37°C is 2.5 – 3.5 μg/ml. SDS-poly-acrylamide gel electrophoresis reveals a degradation of the main protein (150'000 Dalton) into a large fragment (95'000 – 100'000 Dalton) and a small fragment (35'000 – 40'000 Dalton). Increasing ATPase activity correlates with the degree of proteolysis.The _Ca of the digested (Ca²⁺ + Mg²⁺)-ATPase is 0.85 ± 0.1 μM Ca²⁺ as compared to 8.0 ± 0.75 μM Ca²⁺ before digestion and is statistically significantly different from _Ca = 1.66 ± 0.22 μM Ca²⁺ observed in activation by a saturating calmodulin concentration. Addition of calmodulin to the trypsinized enzyme has neither an effect on the Ca²⁺-affinity nor achieves any large increase of the maximal rate.High Ca²⁺ concentrations (above 0.05 – 0.1 mM) after trypsin treatment still inhibit the (Ca²⁺ + Mg²⁺)-ATPase activity. Mg²⁺ activates in the same concentration range ( _Mg = 25 μM) as in the undigested preparation ( _Mg = 27 μM) and retains its competitive behaviour towards Ca²⁺ after trypsin treatment.It is concluded that (1) trypsin treatment unmasks high affinity sites for Ca²⁺ ( _Ca 1 μM) and that, therefore, such sites are not added to the system by calmodulin, and (2) that inhibition by high Ca²⁺-concentrations is not due to Ca - Mg competition at sites located on the calmodulin molecule.     

Cell attachment and spreading on extracellular matrix-coated beads

Parietal yolk sac cells M1536-B3 grown on cytodex 2 beads deposited an extracellular matrix on the surface of the beads. Cell-free matrix-coated beads were isolated by treatment of the cell monolayer with cytochalasin B (CB) at a concentration of 10 μg/ml of phosphate-buffered saline (PBS). The matrix when analysed by electrophoresis on polyacrylamide gels (PAGE) revealed that the major components were laminin and entactin. The matrix-coated beads were used to study the attachment, spreading, and growth of African Green monkey BSC-40, human mammary MCF-7, mouse fibroblast L929, rat liver clone 9, and rat hepatoma H-4-II-E cells in defined serum-free growth medium. The different cell lines exhibited varying responses to matrix-coated vs uncoated beads with respect to rate of attachment, spreading, and growth. One of the most consistent responses observed was the enhancement of cell spreading on matrix-coated beads. The results suggested that the matrix-coated beads will provide a readily available and valuable tool for studies on cell surface-extracellular matrix interactions and the physiological consequences of those interactions.     

Elemental concentrations in plant tissues as influenced by low pH soils

Summary Soils influenced by acid mine drainage (pH<5.0) are characterized by low concentrations of essential nutrients and increased solubility of heavy metals. The conditions typically reduce plant establishment and growth. However, river birch (Betula nigra L.) is commonly found along low pH streams in southeastern Ohio. The objective of this study was to determine the concentration of Al, Mn, Ca and Mg inB. nigra tissues.The results indicate Al and Mn are accumulating inB. nigra when compared to other species. Within river birch, Al concentrations are highest in roots; Mn concentrations are highest in leaves. There is not a concomitant reduction in Ca and Mg concentrations as suggested by soil levels.     

Influence of Manganese on Growth of a Sheathless Strain of Leptothrix discophora

Mn²⁺ exerted various effects on the growth of Leptothrix discophora strain SS-1 in batch cultures depending on the concentration added to the medium. Concentrations of 0.55 to 5.5 μM Mn²⁺, comparable to those in the environment from which strain SS-1 was isolated, decreased cell yield and prolonged stationary-phase survival, but did not affect growth rate. Elevated concentrations of 55 to 910 μM Mn²⁺ also decreased cell yield and prolonged survival, but growth rate was decreased as well. The addition of 1,820 μM Mn²⁺ caused a decline in cell numbers followed by an exponential rise after 80 h of incubation, indicating the development of a population of cells resistant to Mn²⁺ toxicity. When 360 μM Mn²⁺ or less was added to growth flasks, Mn²⁺ was oxidized to manganese oxide (MnO_x, where x is ~2), which appeared as brown particles in the medium. Quantification of Mn oxidation during growth of cultures to which 55 μM Mn²⁺ was added showed that nearly all of the Mn²⁺ was oxidized by the beginning of the stationary phase of growth (15 to 25 h). This result suggested that the decrease in cell yield observed at low and moderate concentrations of Mn²⁺ was related to the formation of MnO_x, which may have bound cationic nutrients essential to the growth of SS-1. The addition of excess Fe³⁺ to cultures containing 55 μM Mn²⁺ increased cell yield to levels near those found in cultures with no added Mn²⁺, indicating that iron deprivation by MnO_x was at least partly responsible for the decreased cell yield.     

Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor

Background

Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.

Methodology/Principal Findings

COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC₅₀ values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D₂ and E₂ in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).

Conclusions/Significance

Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.     

Mechanistic studies on E. coli DNA topoisomerase I: divalent ion effects.

E. coli DNA topoisomerase I catalyzes the hydrolysis of short, single stranded oligodeoxynucleotides. It also forms a covalent protein-DNA complex with negatively supercoiled DNA in the absence of Mg2+ but requires Mg2+ for the relaxation of negatively supercoiled DNA. In this paper we investigate the effects of various divalent metals on catalysis. For the relaxation reaction, maximum enzyme activity plateaus after 2.5 mM Mg2+. However, the rate of cleavage of short oligodeoxynucleotide increased linearly between 0 and 15 mM Mg2+. In the oligodeoxynucleotide cleavage reaction, Ca2+, Mn2+, Co2+, and Zn2+ inhibit enzymatic activity. When these metals are coincubated with Mg2+ at equimolar concentrations, the normal effect of Mg2+ is not detectable. Of these metals, only Ca2+ can be substituted for Mg2+ as a metal cofactor in the relaxation reaction. And when Mg2+ is coincubated with Mn2+, Co2+, or Zn2+ at equimolar concentrations, the normal effect of Mg2+ on relaxation is not detectable. We propose that Mg2+ allows the protein-DNA complex to assume a conformation necessary for strand passage and enhance the rate of enzyme turnover.