A monomeric StayGold fluorescent protein.

StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching. Despite favorable fluorescence properties, use of StayGold as a fluorescent tag is limited because it forms a natural dimer. Here we report the 1.6 Å structure of StayGold and generate a derivative, mStayGold, that retains the brightness and photostability of the original protein while being fully monomeric.

Activator-induced conformational changes regulate division-associated peptidoglycan amidases.

AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from Escherichia coli that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of the cell envelope. In vivo, the activity of AmiA and AmiB is tightly controlled through their interactions with the membrane-bound FtsEX-EnvC complex. Activation of AmiA and AmiB requires access to a groove in the amidase-activating LytM domain of EnvC which is gated by ATP-driven conformational changes in FtsEX-EnvC complex. Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with their cognate activator EnvC.

Insights into bacterial cell division from a structure of EnvC bound to the FtsX periplasmic domain.

FtsEX is a bacterial ABC transporter that regulates the activity of periplasmic peptidoglycan amidases via its interaction with the murein hydrolase activator, EnvC. In Escherichia coli, FtsEX is required to separate daughter cells after cell division and for viability in low-osmolarity media. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for amidase activation, but the process itself is poorly understood. Here we present the 2.1 Å structure of the FtsX periplasmic domain in complex with its periplasmic partner, EnvC. The EnvC-FtsX periplasmic domain complex has a 1-to-2 stoichiometry with two distinct FtsX-binding sites located within an antiparallel coiled coil domain of EnvC. Residues involved in amidase activation map to a previously identified groove in the EnvC LytM domain that is here found to be occluded by a "restraining arm" suggesting a self-inhibition mechanism. Mutational analysis, combined with bacterial two-hybrid screens and in vivo functional assays, verifies the FtsEX residues required for EnvC binding and experimentally test a proposed mechanism for amidase activation. We also define a predicted link between FtsEX and integrity of the outer membrane. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for resistance to membrane-attacking antibiotics and detergents to which E. coli would usually be considered intrinsically resistant. These structural and functional data provide compelling mechanistic insight into FtsEX-mediated regulation of EnvC and its downstream control of periplasmic peptidoglycan amidases.

Association between telomere length and chromosome 21 nondisjunction in the oocyte.

Chromosome 21 nondisjunction in oocytes is the most common cause of trisomy 21, the primary chromosomal abnormality responsible for Down syndrome (DS). This specific type of error is estimated to account for over 90 % of live births with DS, with maternal age being the best known risk factor for chromosome 21 nondisjunction. The loss of telomere length and the concomitant shortening of chromosomes are considered a biological marker for aging. Thus, we tested the hypothesis that mothers who had a maternal nondisjunction error leading to a live birth with DS (n = 404) have shorter telomeres than mothers with live births without DS (n = 42). In effect, our hypothesis suggests that mothers of children with DS will appear "biologically older" as compared to the mothers of euploid children. We applied a quantitative PCR assay to measure the genome-wide relative telomere length to test this hypothesis. The results of our study support the hypothesis that young mothers of DS babies are "biologically older" than mothers of euploid babies in the same age group and supports telomere length as a biomarker of age and hence risk for chromosome nondisjunction.

Metallothionein-3 is a component of a multiprotein complex in the mouse brain.

Metallothionein (MT)-3, originally called growth inhibitory factor (GIF), was initially identified through its ability to inhibit the growth of neuronal cells in the presence of brain extract. MT-3 is the brain specific isoform of the MT family whose specific biological activity associates it with neurological disorders. Indeed, studies report that MT-3 is decreased by ~30% in brains of patients with Alzheimer disease (AD). Furthermore, many lines of evidence suggest that MT-3 engages in specific protein interactions. To address this, we conducted immunoaffinity chromatography experiments using an immobilized anti-mouse MT-3 antibody. We identified five associated proteins from the pool of sixteen recovered using mass spectrometry and tandem mass spectrometry after in-gel trypsin digestion of bands from the affinity chromatography. The proteins identified were: heat shock protein 84 (HSP84), heat shock protein 70 (HSP70), dihydropyrimidinase-like protein-2 (DRP-2), creatine kinase (CK) and beta-actin. Coimmunoprecipitation experiments, also conducted on whole mouse brain extract using the anti-mouse MT-3 antibody along with commercially available antibodies against HSP84 and CK, confirmed that these three proteins were in a single protein complex. Immunohistochemical experiments were then conducted on the perfused mouse brain that confirmed the in situ colocalization of CK and MT-3 in the hippocampus region. These data provide new insights into the involvement of MT-3 in a multiprotein complex, which will be used to understand the biological activity of MT-3 and its role in neurological disease.

Susceptibility of DPOAEs to sound overexposure in inbred mice with AHL.

The notion that three inbred strains of mice, i.e., C57BL/6J (C57), BALB/cByJ (BALB), and WB/ReJ (WB), which exhibit differential rates of age-related hearing loss (AHL), may also exhibit differential susceptibility to noise-induced hearing loss was tested by comparing the effects of sound overexposure on these strains. The aftereffects of noise overstimulation on the distortion-product otoacoustic emissions (DPOAEs) of these three strains were compared and contrasted to those for the CBA/CaJ (CBA) strain, which does not show changes in hearing threshold sensitivity up to 15 months of age. Two cohorts of mice, one at 2.5 and the other at 6 months of age, were first exposed to a tonal overstimulation paradigm, were allowed to recover, and then were later re-exposed to an octave band noise (OBN), at 3 or 7 months of age, respectively. The two sound exposure episodes were designed to produce either a temporary (tonal exposure) or permanent (OBN exposure) reduction in the levels of the 2f1 - f2 DPOAE in the WB strain, which exhibited the fastest rate of AHL. Although the tonal paradigm resulted in a temporary decrease in DPOAE levels for all strains at both ages, the 2.5-month BALBs showed the greatest susceptibility to this overexposure, while the 2.5-month WBs exhibited the least effects on DPOAEs. At the older age of 6 months, tonal overexposure produced essentially the same reduction in DPOAE levels for all four strains. In addition, there were no differences noted between CBAs and C57s, at either of the two ages. The OBN paradigm resulted in a permanent decrease in DPOAE levels in all the strains exhibiting early AHL, i.e., the C57, BALB, and WB mice, for frequencies about one-half to an octave higher than the exposure frequency, regardless of age. In contrast, the CBA strain was not significantly affected by the OBN overexposure.

Temporary and permanent noise-induced changes in distortion product otoacoustic emissions in CBA/CaJ mice.

A number of studies have shown that the ear can be protected from sound over-exposure, either by activating the cochlear efferent system, or by sound 'conditioning' in which the role of the efferent system is less certain. To study more definitively the molecular basis of deliberately induced cochlear protection from excessive sounds, it is advantageous to determine, for an inbred mouse strain, a range of noise exposure parameters that effectively alter cochlear function. As an initial step towards this goal, young CBA/CaJ mice were exposed to a 105-dB SPL octave-band noise (OBN), centered at 10 kHz, for various lengths of time consisting of 10 min, or 0.5, 1, 3, or 6 h. Distortion product otoacoustic emissions (DPOAEs) at the 2f1-f2 frequency, in response to equilevel primary tones of low to moderate levels, were used to quantify the damaging effects of these sound over-exposures on cochlear function. In addition, staining for acetylcholinesterase (AChE) activity to assess for noise-induced changes in the pattern of efferent-nerve innervation to the cochlea was also performed in a subset of mice that were exposed to the longest-lasting 6-h OBN. The 10-min OBN resulted in only temporary reductions in DPOAE levels, which recovered to pre-exposure values within 5 days. Increasing the exposure to 0.5 h resulted in permanent DPOAE losses that, for low primary-tone levels, were still present at 31 days post-exposure. Additionally, the 1-h and longer exposures caused permanent reductions in DPOAEs for all test levels, which were measurable at 31 days following exposure. Light-microscopic observations restricted to the 11-18-kHz frequency region of the organ of Corti, for a subset of mice exposed to the 6-h OBN, uncovered a significant loss of outer hair cells (OHCs). However, despite the OHC loss in this region, the AChE activity associated with the related pattern of efferent innervation remained largely intact.

Identification of the t-type calcium channel (Ca(v)3.1d) in developing mouse heart.

During cardiac development, there is a reciprocal relationship between cardiac morphogenesis and force production (contractility). In the early embryonic myocardium, the sarcoplasmic reticulum is poorly developed, and plasma membrane calcium (Ca(2+)) channels are critical for maintaining both contractility and excitability. In the present study, we identified the Ca(V)3.1d mRNA expressed in embryonic day 14 (E14) mouse heart. Ca(V)3.1d is a splice variant of the alpha1G, T-type Ca(2+) channel. Immunohistochemical localization showed expression of alpha1G Ca(2+) channels in E14 myocardium, and staining of isolated ventricular myocytes revealed membrane localization of the alpha1G channels. Dihydropyridine-resistant inward Ba(2+) or Ca(2+) currents were present in all fetal ventricular myocytes tested. Regardless of charge carrier, inward current inactivated with sustained depolarization and mirrored steady-state inactivation voltage dependence of the alpha1G channel expressed in human embryonic kidney-293 cells. Ni(2+) blockade discriminates among T-type Ca(2+) channel isoforms and is a relatively selective blocker of T-type channels over other cardiac plasma membrane Ca(2+) handling proteins. We demonstrate that 100 micromol/L Ni(2+) partially blocked alpha1G currents under physiological external Ca(2+). We conclude that alpha1G T-type Ca(2+) channels are functional in midgestational fetal myocardium.

Hypaxial muscle migration during primary myogenesis in Xenopus laevis.

In contrast to many vertebrates, the ventral body wall muscles and limb muscles of Xenopus develop at different times. The ventral body wall forms in the tadpole, while limb (appendicular) muscles form during metamorphosis to the adult frog. In organisms that have been examined thus far, a conserved mechanism has been shown to control migratory muscle precursor specification, migration, and differentiation. Here, we show that the process of ventral body wall formation in Xenopus laevis is similar to hypaxial muscle development in chickens and mice. Cells specified for the migratory lineage display an upregulation of pax3 in the ventro-lateral region of the somite. These pax3-positive cells migrate ventrally, away from the somite, and undergo terminal differentiation with the expression of myf-5, followed by myoD. Several other genes are selectively expressed in the migrating muscle precursor population, including neural cell adhesion molecule (NCAM), Xenopus kit related kinase (Xkrk1), and Xenopus SRY box 5 (sox5). We have also found that muscle precursor migration is highly coordinated with the migration of neural crest-derived melanophores. However, by extirpating neural crest at an early stage and allowing embryos to develop, we determined that muscle precursor migration is not dependent on physical or genetic interaction with melanophores.

Nonlinear interactions that could explain distortion product interference response areas.

Suppression and/or enhancement of third- and fifth-order distortion products by a third tone that can have a frequency more than an octave above and a level more than 40 dB below the primary tones have recently been measured by Martin et al. [Hear. Res. 136, 105-123 (1999)]. Contours of iso-suppression and iso-enhancement that are plotted as a function of third-tone frequency and level are called interference response areas. After ruling out order aliasing, two possible mechanisms for this effect have been developed, a harmonic mechanism and a catalyst mechanism. The harmonic mechanism produces distortion products by mixing a harmonic of one of the primary tones with the other primary tone. The catalyst mechanism produces distortion products by mixing one or more intermediate distortion products that are produced by the third tone with one or more of the input tones. The harmonic mechanism does not need a third tone and the catalyst mechanism does. Because the basilar membrane frequency response is predicted to affect each of these mechanisms differently, it is concluded that the catalyst mechanism will be dominant in the high-frequency regions of the cochlea and the harmonic mechanism will have significant strength in the low-frequency regions of the cochlea. The mechanisms are dependent on the existence of both even- and odd-order distortion, and significant even- and odd-order distortion have been measured in the experimental animals. Furthermore, the nonlinear part of the cochlear mechanical response must be well into saturation when input tones are 50 or more dB SPL.

Vasospasm of the internal auditory artery: significance in cerebellopontine angle surgery.

BACKGROUND: Cochlear ischemia is likely involved in sensorineural hearing loss after cerebellopontine angle (CPA) surgery. OBJECTIVE: To demonstrate the type of vascular damage to the cochlea, apart from arterial section, that can be induced by CPA surgery. METHODS: The effects on measures of both cochlear blood flow (CBF) and distortion-product otoacoustic emissions (DPOAEs) of partial or total mechanical compressions of the internal auditory artery (IAA) were compared in young adult rabbits. RESULTS: When preocclusion baseline activity was compared with postocclusion CBF and DPOAEs, it was clear in the majority of cases that total compressions lasting < or =7 minutes produced the same full recoveries for both measures as did the shorter obstructions of only a few minutes. By contrast, both short and long partial occlusions in which ischemia was interrupted by periods of poor reperfusion (<50% of the initial CBF value) resulted in delayed and prolonged recoveries. In addition, at times, full recovery was not achieved, particularly for DPOAEs, because of vasospasm-like activity. CONCLUSION: Vasospasm of the IAA was induced by a systematic series of IAA compressions and releases that did not provide for full reperfusion. These data support the concept that vasospasm should be prevented whenever hearing preservation is attempted in CPA surgery.

Arginine to citrulline replacement in substrates of phosphorylase kinase.

Synthetic peptides based on residues 9 to 18 of glycogen phosphorylase were prepared containing citrulline at position 10 or 16, or at both positions 10 and 16. The peptides were compared as substrates for a recombinant, truncated form of the catalytic subunit of phosphorylase kinase (residues 1-300). The peptide having citrulline at position 10 was phosphorylated the same as the parent peptide. Both the peptides with a single citrulline at position 16 and with two citrullines were phosphorylated less effectively than the parent peptide; k(cat)/K(m) values were approximately 20% the value with the parent peptide. Incorporation of the second citrulline had little change in the effectiveness of the peptide as a substrate although the kinetic parameters with the citrulline peptides did show differences. The change in peptide phosphorylation did not seem to result from a change in peptide structure. Two-dimensional nuclear magnetic resonance studies of di-citrulline peptide are reported and showed no change in the solution structure of the peptide compared to the parent peptide. Thus, the change in kinetic parameters with the modified peptides seemed an effect of arginine replacement and was likely a consequence of the loss of charge inasmuch as the size of arginine and citrulline are similar. Arginine-16 was concluded to be more important for phosphorylase kinase recognition than arginine-10. These findings were consistent with the earlier studies using alanine replacement of arginine in synthetic peptides as substrates for the holoenzyme form of phosphorylase kinase.

In vitro effect of retinoids on calcineurin activity.

Calcineurin was shown previously to be inhibited by members of the tyrphostin family of tyrosine kinase inhibitors, with the most effective inhibition suggested to be caused by the presence of a conjugated side chain (Martin BL, Biochem Pharmacol 56: 483-488, 1998). Retinoids are a family of naturally occurring biomolecules having non-aromatic ring structures and conjugated side chains as substituents on the ring. Three oxidation states of the all-trans configuration of retinoids (retinol, retinal, and retinoic acid) were tested as effectors of calcineurin. Only retinoic acid was found to inhibit calcineurin effectively, with an IC(50) value of approximately 50 microM. Retinol and retinal caused less than 30% inhibition at concentrations up to 100 microM. All three retinoids caused some precipitation of reaction components: retinoic acid and retinal above 50 microM, and retinol above 250 microM. Bacterial alkaline phosphatase was not inhibited by the retinoids, indicating that metal centers alone are insufficient for significant inhibition by retinoic acid. An aromatic ring was not required for inhibition and may not provide additional inhibition, inasmuch as an aromatic analog of retinoic acid (acitretin) showed less effective inhibition. These data are consistent with the presence of conjugated, unsaturated groups enhancing the inhibition of calcineurin.

Differences between Mg(2+) and transition metal ions in the activation of calcineurin.

Exogenous metal ion activation of calcineurin catalyzed hydrolysis of para-nitrophenyl phosphate was kinetically characterized at 20, 25, 30, and 37 degrees C. Analysis yielded estimates for thermodynamic parameters for the activation of calcineurin by each of the metal ions. Values for DeltaG(Me)( degrees ) were varied with the best activators resulting in more stable enzyme-metal ion complexes and with DeltaG(Me)( degrees ) dominated by the entropic component. Mg(2+) was the only nontransition metal ion which supported significant activity and showed some distinct characteristics including a negative DeltaS(Me)( degrees ), suggesting that activation by Mg(2+) may have resulted in a unique enzyme-metal ion form. Circular dichroism showed that metal ions increased the alpha-helical content of calcineurin, but little significant differences in the spectra were identified between using activating and nonactivating metal ions. Activating Mg(2+), but not nonactivating Ca(2+), did cause changes in the Fourier transform infrared photoacoustic spectrum of calcineurin compared to the spectrum of calcineurin with Mn(2+). Other metal ions, Co(2+) and Ni(2+), also caused no changes in the infrared spectrum. Possible explanations for these differences between Mg(2+) and transition metal ions in the activation of calcineurin are discussed.

Effects of cis-platinum chemotherapy on otoacoustic emissions: the development of an objective screening protocol. Third place--Resident Clinical Science Award 1998.

To develop an objective, fast, and simply performed screening protocol for cis -platinum (CP) ototoxicity, we compared the efficacy of screening with distortion-product otoacoustic emissions (DPOAEs) with the outcome of both conventional and ultra-high-frequency (UHF) audiometry. Baseline audiometric and DPOAE testing was performed in 66 patients, 33 of whom met criteria for inclusion in the final database. Comparisons were made between baseline measurements and those recorded before subsequent CP infusions. Outcomes were analyzed clinically and with paired repeated-measures analysis of variance. Results indicated that DPOAEs and UHF were better measures than conventional audiometry. Further, DPOAEs may be better suited for screening older patients receiving CP chemotherapy because DPOAEs are as sensitive as UHF and are present in a greater number of these patients. Screening with DPOAEs may be enhanced by testing only in the 3- to 5.2-kHz range, thus decreasing testing time. Higher time averages to increase the signal-to-noise ratio and use of this narrower bandwidth might also allow for accurate bedside testing.

Age-related loss of distortion product otoacoustic emissions in four mouse strains.

Changes in cochlear function in four inbred strains of mice, CBA/CaJ (CBA), C57BL/6J (C57), BALB/cByJ (BALB), and WB/ReJ (WB), previously used to study age-related hearing loss, were evaluated serially as a function of age with 2f(1)-f(2) distortion-product otoacoustic emissions (DPOAEs). DPOAE levels in response to equilevel primary tones for geometric-mean (GM) frequencies from 5.6 to 48.5 kHz were recorded systematically as DP-grams and response/growth or input/output (I/O) functions at monthly intervals from about 2 to 15 months of age. Over the approximate 13-month measurement period, CBAs showed robust and unchanged DPOAEs for all tested frequencies, while BALBs, C57s, and WBs showed strain-specific, age-related decreases in DPOAEs that progressed systematically from the high to low frequencies. Specifically, for the youngest WBs at 2 months of age, no DPOAEs were recordable for GM frequencies > or = 32 kHz, while C57s and BALBs reached the identical stage of cochlear dysfunction by 5 and 8 months, respectively. The differential decline in DPOAE activity shown for WB, C57, and BALB mice supports the notion that they represent unique animal models of age-related changes in cochlear function. In contrast, the unchanging DPOAEs for CBAs over the same time period indicate that this strain makes an effective control for normal cochlear function in the mouse, at least, up to 15 months of age.

Suppression and enhancement of distortion-product otoacoustic emissions by interference tones above f(2). I. Basic findings in rabbits.

The present study measured interference-response areas (IRAs) for distortion-product otoacoustic emissions (DPOAEs) at 2f(1)-f(2), 3f(1)-2f(2), and 2f(2)-f(1). The IRAs were obtained in either awake or anesthetized rabbits, or in anesthetized guinea pigs and mice, by sweeping the frequencies and levels of an interference tone (IT) around a set of f(1) and f(2) primary tones, at several fixed frequencies and levels, while plotting the effects of the IT on DPOAE level. An unexpected outcome was the occurrence of regions of suppression and/or enhancement of DPOAE level when the IT was at a frequency slightly less than to more than an octave above f(2). The IRA of the 2f(1)-f(2) DPOAE typically displayed a high-frequency (HF) lobe of suppression, while the 2f(2)-f(1) emission often exhibited considerable amounts of enhancement. Moreover, for the 2f(2)-f(1) DPOAE, when enhancement was absent, its IRA usually tuned to a region above f(2). Whether or not suppression/enhancement was observed depended upon primary-tone level and frequency separation, as well as on the relative levels of the two primaries. Various physiological manipulations involving anesthesia, eighth-nerve section, diuretic administration, or pure-tone overstimulation showed that these phenomena were of cochlear origin, and were not dependent upon the acoustic reflex or cochlear-efferent activity. The aftereffects of applying diuretics or over-exposures revealed that suppression/enhancement required the presence of sensitive, low-level DPOAE-generator sources. Additionally, suppression/enhancement were general effects in that, in addition to rabbits, they were also observed in mice and guinea pigs. Further, corresponding plots of DPOAE phase often revealed areas of differing phase change in the vicinity of the primary tones as compared to regions above f(2). These findings, along with the effects of tonal exposures designed to fatigue regions above f(2), and instances in which DPOAE level was dependent upon the amount of suppression/enhancement, suggested that the interactions of two DPOAE-generator sources contributed, in some manner, to these phenomena.

Measuring the cochlear blood flow and distortion-product otoacoustic emissions during reversible cochlear ischemia: a rabbit model.

Impairment to the cochlear blood flow likely induces many types of sensorineural hearing loss. Models using several small laboratory animals have been described in the literature that permit the simultaneous monitoring of the cochlear blood flow with laser-Doppler flowmetry and cochlear function using evoked responses. However, these models have not permitted a direct application of the resulting knowledge to the human condition, primarily due to differences in the translucence of the otic capsule between species. In the present study, to approximate conditions relevant to the human patient, the rabbit was utilized to develop a procedure in which laser-Doppler flowmetry could be used to measure the cochlear blood flow in an animal with an opaque otic capsule. At the same time, the cochlear function was monitored non-invasively using distortion-product otoacoustic emissions. In this manner, a laser-Doppler probe was positioned in the round window niche and the cochlear function measured using distortion-product otoacoustic emissions during a systematic series of ischemic episodes. Cochlear ischemia was produced by deliberately compressing the eighth nerve complex at the porus of the internal acoustic meatus, for periods lasting from 1-3 min, while cochlear blood flow and distortion-product otoacoustic emission measures were obtained simultaneously before, during and following the occlusion. Results demonstrated that the cochlear blood flow sharply decreased within 1 s after compression onset, whereas distortion-product otoacoustic emissions showed obstruction-induced changes after a delay of several seconds, provided that the blood flow decreased, at least 40%. Similarly, upon release of the compression, the cochlear blood flow began to recover within 1 s, whereas the recovery of the corresponding distortion-product otoacoustic emissions was slightly delayed. Although not apparent in the distortion-product otoacoustic emission recovery time course, the cochlear blood flow consistently overshot its initial baseline value during the recovery process. Thus, although cochlear ischemia produced changes in the distortion-product otoacoustic emission activity that generally followed the resulting alterations in the cochlear blood flow, the detailed relationship between the two measures was complex.

Effect of substitution inert metal complexes on calcineurin.

As a substitute for M(H2O)2+6, Co(NH3)3+6 was found to activate calcineurin with para-nitrophenyl phosphate as substrate. Kinetics for calcineurin catalyzed hydrolysis of para-nitrophenyl phosphate at pH 7.0 with Mn2+, Mg2+, Co2+, and Co(NH3)3+6 were compared. Although kcat and Km were different with the metals, values of kcat/Km were nearly identical for Mn2+ and Mg2+, but lower for Co2+ and Co(NH3)3+6. The concentration of each metal providing half-maximal activation, designated Kact, was evaluated as 15.9 mM for Co(NH3)3+6, compared to Kact = 0.17 mM for Mn2+ and Co2+ and 6.3 mM for Mg2+, respectively. Comparing kcat/Kcat showed that Co(NH3)3+6 was a 170-fold poorer activator of calcineurin than was Mn2+, but only 1.5-fold poorer than Mg2+. Activation by Co(NH3)3+6 indicated that activation of calcineurin by exogenous metal ions can proceed via an outer coordination sphere reaction mechanism with no requirement for the direct coordination of substrate by metal. Because Co(NH3)3+6 was found to support calcineurin activity, the related compound [Co-(ethylenediamine)3]3+ (or Co(en)3+3) was tested as a possible activator. Co(en)3+3 did not support calcineurin activity but did inhibit calcineurin. Co(en)3+3 showed competitive inhibition kinetics with either Mn2+ or pNPP as the varied ligand and the other at a fixed, subsaturating concentration. Inorganic phosphate was used as a known competitive inhibitor to pNPP (B. L. Martin and D. J. Graves, J. Biol. Chem. 261, 14545-14550, 1986) and showed uncompetitive inhibition with Mn2+ as the varied ligand. These patterns are consistent with the mechanism of ligand binding to calcineurin being ordered with metal preceding substrate. Prior formation of a metal-substrate complex was not required for association with calcineurin.