Binding of polonium-210 to liver metallothionein

The interactions of 210Po at the molecular level in biological systems have received little study even though this alpha-emitting radionuclide occurs widely in nature. Polonium-210 was given subcutaneously to rats and found to be incorporated into liver metallothionein as judged by a number of criteria including heat stability, acetone precipitation, and chromatography. In vitro studies confirmed this binding. The binding of 210Po to metallothionein has implications that may help explain some of the radiation damage 210Po causes intracellularly.     

Metal selectivity of clusters in rabbit liver metallothionein.

In mammalian metallothioneins the metals are organized in two adamantane-type clusters with three and four metal ions which are tetrahedrally coordinated by thiolate ligands. The metal selectivity of the metal-thiolate clusters in rabbit liver metallothionein has been studied by offering two ions, i.e. Co(II)/Cd(II), Zn(II)/Cd(II) or Co(II)/Zn(II), to the metal-free protein. The heterogeneous metal complexes thus formed were characterized by electronic absorption, magnetic circular dichroism. 113Cd-NMR and EPR spectroscopy. In the case of Co/Cd-metallothionein, homometallic cluster occupation occurs, with the Cd(II) ions bound exclusively to the four-metal cluster. In contrast, heterometallic clusters were formed for both Zn/Cd- and Co/Zn-metallothionein. Based on evidence from corresponding inorganic structures of adamantane metal-thiolate cages, it is suggested that the major factor governing the cluster type is the protein structure perturbation due to the cluster volume variations. Thus, while metal thiolate affinities are important in the folding process, size-match selectivity is the dominant factor in the metal-loaded protein.     

Gram scale purification and preparation of rabbit liver zinc metallothionein.

A simplified procedure for purifying gram quantities of rabbit liver metallothionein (MT) using gel filtration and anion exchange chromatography is presented. The MT purification made use of anion exchange batch elution chromatography which greatly shortened the procedure. Quantitation techniques for use with crude and purified MT are discussed. This paper also describes the preparation of large amounts of ZnMT from Cd,ZnMT.     

Characterization of metallothionein isoforms from rabbit liver by liquid chromatography coupled to electrospray mass spectrometry

Metallothioneins (MTs), a group of low molecular weight proteins found in practically all life forms, are characterized by high sulfur content and an affinity for metal ions. At acidic pH, MTs show metal depletion, leading to apothioneins. In the work described here, in order to optimize the separation of rabbit liver apothioneins using liquid chromatography (LC) with UV detection, the proportion of the organic modifier of the mobile phase was optimized by establishing relationships between Reichardt's E(N)(T) scale of solvent polarity and the chromatographic retention measured by the capacity factor, k. Additionally, such optimum separations were carried out in a LC-electrospray mass spectrometry (ES-MS) coupled system allowing the identification and characterization of the different rabbit liver apo-MT-forms. In this way, electrospray ionization mass spectrometry offers great possibilities aiming at a better understanding of metallothionein polymorphism.     

Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (Pi) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with Pi, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when Pi is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.     

The effect of inorganic phosphate on force generation in single myofibrils from rabbit skeletal muscle

In striated muscle, force generation and phosphate (P(i)) release are closely related. Alterations in the [P(i)] bathing skinned fibers have been used to probe key transitions of the mechanochemical coupling. Accuracy in this kind of studies is reduced, however, by diffusional barriers. A new perfusion technique is used to study the effect of [P(i)] in single or very thin bundles (1-3 microM in diameter; 5 degrees C) of rabbit psoas myofibrils. With this technique, it is possible to rapidly jump [P(i)] during contraction and observe the transient and steady-state effects on force of both an increase and a decrease in [P(i)]. Steady-state isometric force decreases linearly with an increase in log[P(i)] in the range 500 microM to 10 mM (slope -0.4/decade). Between 5 and 200 microM P(i), the slope of the relation is smaller ( approximately -0.07/decade). The rate constant of force development (k(TR)) increases with an increase in [P(i)] over the same concentration range. After rapid jumps in [P(i)], the kinetics of both the force decrease with an increase in [P(i)] (k(Pi(+))) and the force increase with a decrease in [P(i)] (k(Pi(-))) were measured. As observed in skinned fibers with caged P(i), k(Pi(+)) is about three to four times higher than k(TR), strongly dependent on final [P(i)], and scarcely modulated by the activation level. Unexpectedly, the kinetics of force increase after jumps from high to low [P(i)] is slower: k(Pi(-)) is indistinguishable from k(TR) measured at the same [P(i)] and has the same calcium sensitivity.     

Nuclear magnetic resonance studies of inorganic phosphate binding to yeast inorganic pyrophosphatase.

Yeast inorganic pyrophosphatase is a dimer of identical subunits. Previous work (Rapoport, T.A., et al. (1973) Eur. J. Biochem. 33, 341) indicated the presence of two different Mn2+ binding sites per subunit. In the present work, the binding of inorganic phosphate to the Mn2+-inorganic pyrophosphatase complex has been studied by 1H and 31P nuclear magnetic resonance. Two distinct phosphate sites have been found, having dissociation constants of 0.24 mM and 18 mM. The Mn2+-31P distance from tightly bound Mn2+ to phosphate bound in the low affinity site (6.2 A) is consistent with outer sphere binding. Binding to both phosphate sites can be simultaneously inhibited by the pyrophosphate analogue, hydroxymethanebisphosphonate, providing evidence for the physical proximity of these two sites. The weaker Mn2+ site is apparently far from both phosphate sites. From the magnitudes of the dissociation constants found for both phosphate and analogue binding and the recent work of P.D. Boyer and his co-workers (private communication) on enzyme-catalyzed phosphate-water exchange, it appears unlikely that the hydrolysis of enzyme-bound pyrophosphate is the rate-determining step in the overall enzymatic catalysis of pyrophosphate hydrolysis, at least when Mn2+ is the required divalent metal ion cofactor.     

Importance of storage conditions for the stability of zinc- and cadmium-induced metallothionein

We examined the storage stability of metallothionein (MT), a cysteine-rich protein that has diagnostic potential as a cancer marker and in the assessment of Zn status and heavy-metal toxicity. MT was rapidly degraded in samples of rat whole liver at -20 degrees C or -70 degrees C. MT in supernatants from heat-treated rat liver homogenates stored as 1:5 dilutions of liver from Zn- or Cd-induced rats were stable (recovery >98%) for 100 d at temperatures of -70 degrees C and -196 degrees C but not at -20 degrees C, regardless of the presence of dithiothreitol (DTT) or argon. The variability of MT measurement by the 109Cd-hemoglobin affinity assay was however greatest in samples from Zn-induced rats stored without DTT. The integrity of the MT protein in supernatants of heat-treated homogenates stored for 100 d was demonstrated by Sephadex G-75 chromatography. When heat-treated supernatants were stored as dilute solutions (1:125 of liver), MT was unstable regardless of treatment or storage temperature. Our findings show that liver MT is stable for at least 4 mo as a supernatant of a heat-treated homogenate (1:5 dilution of liver) when stored at or below -70 degrees C and in the presence of DTT.     

Isolation and partial characterization of native metallothionein in fetal rabbit liver

The endogenous zinc content of hepatic cytosol in the term fetal rabbit was found to be three-fold higher than corresponding maternal levels. Following gel-filtration, the bulk of this zinc was found to be associated with a peak, not detectable in the maternal cytosol, having a relative elution volumn similar to that of cadmium-induced metallothionein. $\text{In vitro}$ addition of increasing amounts of cadmium to the fetal cytosol prior to gel-filtration caused progessive displacement of endogenous zinc from the peak, selective cadmium binding and eventual cadmium saturation. Anion-exchange chromatography disclosed two distinct peaks, corresponding to the two cadmium-containing peaks detected in the cytosol from cadmium-induced animals. SDS-polyacrylamide gel electrophoresis of purified protein also revealed the existance of two bands in both the fetus and induced adult. Results indicate the presence of endogenous metallothioneins in the hepatic cytosol of the normal term rabbit fetus which are similar to the metallothioneins which appear in the adult hepatic cytosol following exposure to cadmium.     

Phosphorylation of pyruvate kinase type K is restricted to the dimeric form.

In the absence of glycolytic intermediate, fructose-1,6-bisphosphate, pyruvate kinase type K exists in the dimeric form and is readily phosphorylated, whereas in the same sample and the same conditions pyruvate kinase type M is present as a tetramer and is not phosphorylated. Addition of fructose-1,6-bisphosphate results in the association of dimeric K2 molecules to a tetrameric K4 enzyme as determined by gel filtration and cellulose acetate electrophoresis, with concomitant loss of the capacity of the K isozyme to become phosphorylated. Phosphorylated K2 dimers can also tetramerize, but with a low recovery of the radiolabel, suggesting a fructose-1,6-bisphosphate induced dephosphorylation or selective degradation. The dimeric K isozyme is enzymatically active; inactive K-type monomers can be detected by immunoblot analysis in the absence of fructose-1,6-bisphosphate, but no phosphorylated pyruvate kinase is present in this fraction. The formation of K4 tetramers can not be accomplished by the substrate phosphoenolpyruvate. Fructose-1,6-bisphosphate is an allosteric activator of pyruvate kinase type K and induces hyperbolic saturation curves for phosphoenolpyruvate. In contrast, in the absence of effectors, pyruvate kinase type M exhibits Michaelis-Menten kinetics, but sigmoidal curves can be induced by the amino acid phenylalanine. However, even in the presence of phenylalanine, the M-type maintained its tetrameric configuration and did not serve as a substrate in the phosphorylation reaction. These findings argue for the importance of subunit interaction in the regulation of phosphorylation of pyruvate kinase.     

Stabilization of active form of rabbit liver phosphofructokinase

(1) The active form of rabbit liver phosphofructokinase when preincubated in presence of F^? and effectors of the enzyme is stabilized against its conversion to less active form as a result of dilution. (2) The stabilized active form of enzyme has a Km value of 0.01 mM for fructose 6-phosphate, the same as measured in presence of all the positive effectors, and is lower, by 13 times, than the Km value of the non-stabilized control enzyme, and exhibits normal Michaelis-Menten kinetics, in contrast to the non-stabilized control enzyme which shows sigmoidal kinetics. (3) The stabilized active form of enzyme is neither inhibited by excess concentration of ATP nor activated by activators of phosphofructokinase. (4) The data thus support the proposition that the enzyme does indeed exist in two interconvertible forms with enormous difference in their affinities for fructose 6-phosphate and effectors.     

Biochemical characterization of a truncated form of CYP27A purified from rabbit liver mitochondria.

During purification of CYP27A from rabbit liver mitochondria, a cytochrome P450 of different molecular size was co-isolated. The latter enzyme has an apparent M(r) 51,000 which is slightly lower than that of CYP27A. The 51,000-M(r) protein was found to be present in mitochondria from liver, small intestine, kidney, and spleen but not in lung, testis, heart, or brain mitochondria. Determination of the N-terminal sequence revealed that the 51,000-M(r) protein is a truncated form of CYP27A lacking the first 12 residues. The truncated enzyme was less efficient than the full-length CYP27A in the 27-hydroxylation of C(27)-sterols and much less efficient in the 25-hydroxylation of 1alpha-hydroxyvitamin D(3). The K(m) values for cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol were about the same with both enzymes whereas the K(m) for 1alpha-hydroxyvitamin D(3) was much higher with the truncated CYP27A. The results strongly indicate that the 51,000-M(r) protein is formed via proteolytic processing of CYP27A by endogenous protease(s) in some of the tissues examined. The truncation at the N terminus markedly impairs the ability of CYP27A to use 1alpha-hydroxyvitamin D(3) as substrate and to catalyze 25-hydroxylation in the bioactivation of vitamin D(3).     

Binding of dihydrotestosterone to a nuclear-envelope fraction from the male rat liver.

Intact nuclear 'ghosts' containing small amounts of DNA were obtained from rat liver. Incubation of radiolabelled dihydrotestosterone with isolated nuclear-envelope fraction from male rat liver resulted in specific binding of the dihydrotestosterone to the membranes. Optimal binding occurred at 20 degrees C after 20h incubation. Storage for 2 weeks at -80 degrees C resulted in little loss of specific binding. Scatchard analysis revealed a class of binding sites with a KD of 23.2 nM. Pronase and heat treatment destroyed the binding site. Androgens and glucocorticoids competed for labelled dihydrotestosterone binding to the ghosts, whereas oestrogens did not compete. Castration 24h before preparation of ghosts did not alter the binding site, and a similar class of binding sites was identified on female rat liver nuclear envelopes.     

Binding of alkaline cations to the double-helical form of gramicidin.

Gramicidin is a polypeptide antibiotic that forms monovalent cation-specific channels in membrane environments. In organic solvents and in lipids containing unsaturated fatty acid chains, it forms a double-helical "pore" structure, in which two monomers are intertwined. This form of gramicidin can bind two cations inside its lumen, and the crystal structures of both an ion complex and an ion-free form have been determined. In this study, we have used circular dichroism (CD) spectroscopy to examine the binding mechanism and the binding constants (K1 and K2) of cations to gramicidin in the double helical form in methanol solution. The dramatic change in optical rotation in the far-ultraviolet CD spectrum of gramicidin provides a useful tool for monitoring the binding. The binding mechanism appears to involve a large conformation change associated with the binding of ions to the first of the two sites. The calculated values for the K1 binding constants for alkaline cations are considerably smaller than the K2 binding constants. The order of binding affinity for alkaline cations is similar to that for the helical dimer "channel" form of gramicidin, i.e., Cs+ approximately Rb+ > > K+ > Li+, but in comparison to the helical dimer form, the binding to double-helical dimers is dominated by a cation size-dependent conformational change in the gramicidin structure.