Effects of marginal zinc deficiency on microtubule polymerization in the developing rat brain

One of the possible mechanisms that has been proposed to underlie the deleterious effects of zinc deficiency on brain development is an impairment in the normal formation of the cytoskeletal network. In the current study, in vivo microtubule polymerization was characterized in brain supernatant fluids, from 20-d-old pups whose dams were fed diets containing control (50 micrograms zinc/g) or marginal levels of zinc (10 micrograms zinc/g) throughout pregnancy and lactation. Pup brain and body weights were similar between the groups; however, plasma zinc concentrations were lower (27%) in pups fed the marginal zinc diet than in controls. Tubulin concentrations in 100,000 g brain supernates were similar between the groups; however, tubulin polymerization in the brain supernates was significantly lower in pups fed the marginal zinc diet compared to controls. Primarily, the early events of polymerization were affected; the lag period of the reaction was doubled, and the initial velocity was slower (26%) in supernates from pups fed the marginal zinc diet than in controls. These findings support the idea that some of the negative effects of marginal zinc deficiency on brain development and function may be mediated by an alteration in microtubule formation.     

Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization

Clonal cells (N18) of the mouse neuroblastoma C-1300 can be induced to undergo a morphological differentiation characterized by the outgrowth of very long neurites (> 150 microns) that contain many microtubules. Because the marked increase in the number and length of microtubules is apparently not due to an increase in the concentration of tubulin subunits, the possible role of additional macromolecules in the regulation of tubulin polymerization during neurite formation by N18 cells was examined. Using an in vitro system where the polymerization of low concentrations (< 4 mg/ml) of purified brain tubulin requires microtubule-associated proteins (MAPs), high-speed supernates (250,000 g) from neuroblastoma and glioma cells were assayed for their ability to replace MAPs in the polymerization of brain tubulin. Only the supernates from "differentiated" N18 cells were polymerization competent. Electron microscope observations of these supernates failed to demonstrate the presence of nucleation structures (rings or disks). The active factor(s) sedimented at approximately 7S on sucrose gradient centrifugation and eluted from 4B Sepharose in the region of 170,000 mol wt proteins. Furthermore, the inactive supernates from other cells did not inhibit polymerization when tested in the presence of limiting MAPs. Thus, microtubule formation accompanying neurite outgrowth in neuroblastoma cells appears to be regulated by the presence of additional macromolecular factor(s) that may be functionally equivalent to the MAPs found with brain microtubules.     

Susceptibility to kainate-induced seizures under dietary zinc deficiency

Zinc homeostasis in the brain is altered by dietary zinc deficiency, and its alteration may be associated with the etiology and manifestation of epileptic seizures. In the present study, susceptibility to kainate-induced seizures was enhanced in mice fed a zinc-deficient diet for 4 weeks. When Timm's stain was performed to estimate zinc concentrations in synaptic vesicles, Timm's stain in the brain was attenuated in the zinc-deficient mice. In rats fed the zinc-deficient diet for 4 weeks, susceptibility to kainate-induced seizures was also enhanced. When the release of zinc and neurotransmitters in the hippocampal extracellular fluid of the zinc-deficient rats was studied using in vivo microdialysis, the zinc concentration in the perfusate was less than 50% of that of the control rats and the increased levels of zinc by treatment with kainate were lower than the basal level in control rats, suggesting that vesicular zinc is responsive to dietary zinc deficiency. The levels of glutamate in the perfusate of the zinc-deficient rats were more increased than in the control rats, whereas the levels of GABA in the perfusate were not at all increased in the zinc-deficient rats, unlike in the control rats. The present results demonstrate an enhanced release of glutamate associated with a decrease in GABA concentrations as a possible mechanism for the increased seizure susceptibility under zinc deficiency.     

Effects of dietary zinc deficiency on homocysteine and folate metabolism in rats

In rats, zinc deficiency has been reported to result in elevated hepatic methionine synthase activity and alterations in folate metabolism. We investigated the effect of zinc deficiency on plasma homocysteine concentrations and the distribution of hepatic folates. Weanling male rats were fed ad libitum a zinc-sufficient control diet (382.0 nmol zinc/g diet), a low-zinc diet (7.5 nmol zinc/g diet), or a control diet pair-fed to the intake of the zinc-deficient rats. After 6 weeks, the body weights of the zinc-deficient and pair-fed control groups were lower than those of controls, and plasma zinc concentrations were lowest in the zinc-deficient group. Plasma homocysteine concentrations in the zinc-deficient group (2.3 +/- 0.2 micromol/L) were significantly lower than those in the ad libitum-fed and pair-fed control groups (6.7 +/- 0.5 and 3.2 +/- 0.4 micromol/L, respectively). Hepatic methionine synthase activity in the zinc-deficient group was higher than in the other two groups. Low mean percentage of 5-methyltetrahydrofolate in total hepatic folates and low plasma folate concentration were observed in the zinc-deficient group compared with the ad libitum-fed and pair-fed control groups. The reduced plasma homocysteine and folate concentrations and reduced percentage of hepatic 5-methyltetrahydrofolate are probably secondary to the increased activity of hepatic methionine synthase in zinc deficiency.     

Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine

At metaphase, the amount of tubulin assembled into spindle microtubules is relatively constant; the rate of tubulin association equals the rate of dissociation. To measure the intrinsic rate of dissociation, we microinjected high concentrations of colchicine, or its derivative colcemid, into sea urchin embryos at metaphase to bind the free tubulin, thereby rapidly blocking polymerization. The rate of microtubule disassembly was measured from a calibrated video signal by the change in birefringent retardation (BR). After an initial delay after injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM, BR decreased rapidly and simultaneously throughout the central spindle and aster. Measured BR in the central half-spindle decreased exponentially to 10% of its initial value within a characteristic period of approximately 20 s; the rate constant, k = 0.11 +/- 0.023 s-1, and the corresponding half-time, t 1/2, of BR decay was approximately 6.5 +/- 1.1 s in this concentration range. Below 0.1 mM colchicine or colcemid, the rate at which BR decreased was concentration dependent. Electron micrographs showed that the rapid decrease in BR corresponded to the disappearance of nonkinetochore microtubules; kinetochore fiber microtubules were differentially stable. As a control, lumicolchicine, which does not bind to tubulin with high affinity, was shown to have no effect on spindle BR at intracellular concentrations of 0.5 mM. If colchicine and colcemid block only polymerization, then the initial rate of tubulin dissociation from nonkinetochore spindle microtubules is in the range of 180-992 dimers per second. This range of rates is based on k = 11% of the initial polymer per second and an estimate from electron micrographs that the average length of a half-spindle microtubule is 1- 5.5 micron. Much slower rates of tubulin association are predicted from the characteristics of end-dependent microtubule assembly measured previously in vitro when the association rate constant is corrected for the lower rate of tubulin diffusion in the embryo cytoplasm. Various possibilities for this discrepancy are discussed.     

The effect of zinc deficiency and food restriction on prostaglandin E2 and thromboxane B2 in saliva and plasma of rats

Zinc has been implicated in the regulation of prostaglandins and other arachidonic acid derivatives. Studies of zinc-deficient animals, however, are compromised by concomitant reduction in food intake that may also alter eicosanoid levels in body tissues and fluids. In this study, three groups of rats, designated as zinc-deficient, pair-fed and control, were fed diets containing 1 ppm, 15 ppm (in amounts paired to deficient rats) and 15 ppm Zn ad libitum, respectively, for 6 weeks. Saliva and blood were analyzed for PGE2 and TXB2 by radioimmunoassay. Saliva concentrations of both eicosanoids were lower (p less than 0.05) in the pair-fed animals, but not significantly altered by zinc deficiency. Plasma levels of PGE2 and TXB2 were unchanged by either zinc deficiency or food restriction. The results of this study support the contention that the effect of zinc on these prostaglandins is not mediated by altered rates of synthesis or degradation but rather by effects on eicosanoid function.     

Colchicine-binding protein of the liver. Its characterization and relation to microtubules

Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(- 3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time- dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.     

Induction of polymerization of purified tubulin by sulfonate buffers. Marked differences between 4-morpholineethanesulfonate (Mes) and 1,4-piperazineethanesulfonate (Pipes)

Interactions of both purified tubulin and microtubule protein (tubulin plus associated proteins) with two commonly used sulfonate buffers were examined. 1,4-Piperazineethanesulfonate (Pipes) and 4-morpholineethanesulfonate (Mes) at high concentrations induce the polymerization of purified tubulin in reactions requiring only buffer, tubulin and GTP. While both reactions were temperature-dependent, cold-reversible and inhibited by GDP, colchicine or Ca2+, there were significant differences between them. Substantially lower tubulin and buffer concentrations were required for Pipes-induced polymerization; and turbidity was much more intense in the Pipes-induced than in the Mes-induced reaction at the same protein concentration. Electron microscopy demonstrated that for the most part typical smooth-walled microtubules were formed in Mes, while aberrant forms were the predominant structures formed in Pipes. When the polymerization of microtubule protein was examined as a function of buffer concentration, biphasic patterns were observed with both Pipes and Mes: polymerization occurred at both low and high, but not intermediate, buffer concentrations. The turbidity observed at high concentrations of Pipes greatly exceeded that at low concentrations. With Mes, equivalent turbidity developed at both high and low buffer concentrations. Although associated proteins copolymerized with tubulin at low buffer concentrations, they were excluded from the polymerized material at high buffer concentrations. Pipes and Mes were compared to sodium phosphate, Tris/HCl and imidazole/HCl buffers at 0.1 M in several polymerization systems using both purified tubulin and microtubule protein. The sulfonate buffers were invariably associated with more vigorous reactions than the other buffers.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

Interaction of microtubule proteins with phospholipid vesicles

We have examined the interaction of unilamellar dimyristoyl phosphatidylcholine liposomes with the high-speed supernate of brain homogenate and with tubulin purified through one or two cycles of microtubule assembly-disassembly. Tubulin and certian high molecular weight proteins are selectively adsorbed from these mixtures onto liposomes. The composition of adsorbed proteins is similar to that obtained during corresponding cycles of microtubule assembly, suggesting the equivalency of these processes. Adsorption induces stacking and/or fusion of liposomes into multilamellar structures indicating strong protein-lipid interaction. In addition, liposome-adsorbed tubulin forms extensive intermolecular disulfide bridges that are inert to reducing agents in the aqueous medium. The observations form a basis for further study of the distribution, function, and properties of membrane-bound tubulin.     

Effect of dietary zinc intake on the hematological profile of the rat

The effect of dietary zinc deficiency (imposed at weaning) on the hematological profile of the male rat was studied. Lack of dietary zinc resulted in elevated numbers of erythrocytes and segmented neutrophils and decreased reticulocyte number. The hematocrit of zinc-deficient rats was significantly elevated after the first week. The mean lifespan of erythrocytes from zinc-deficient rats (tested in adult control rats) was not significantly altered from that of controls. The lack of dietary zinc did not cause a major change in the erythrocyte density (age) distribution.     

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.     

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.     

Glutamine synthetase cascade: enrichment of uridylyltransferase in Escherichia coli carrying hybrid ColE1 plasmids

Colchicine-binding properties of the total cytoplasmic pool of tubulin from rat liver were evaluated in tubulin-stabilizing (TS) supernates. Microtubules were separated from free tubulin using a microtubule-stabilizing solution (MTS) and ultracentrifugation. [³H]Colchicine-binding properties of microtubule-derived tubulin were investigated in supernates prepared after resuspension of MTS pellets in TS. In TS buffer at 37 °C the colchicine-binding activity of the total cytoplasmic pool of tubulin decayed with $\text{T}\text{1}\text{2}$ of 3.39 h. Resuspended pellet tubulin decayed much more rapidly under the same conditions with a $\text{T}\text{1}\text{2}$ of 0.72 h. This rapid time decay of microtubule-derived tubulin was found to be at least partially attributable to prior microtubule-stabilizing solution exposure. Since tartrate has been reported to increase the rate of colchicine binding to tubulin, sodium tartrate (150 mm) was added to our colchicine-binding system. This addition increased the detectable [³H]colchicine binding by 10% in the total cytoplasmic preparation and by 85% in the resuspended pellet preparation. Addition of tartrate (150 mm) also resulted in a 105% increase in the $\text{T}\text{1}\text{2}$ for total cytoplasmic tubulin and a 412% increase for microtubule derived tubulin. Total cytoplasmic supernates of liver bound [³H]colchicine linearly over a wide range of tissue concentrations. However, resuspended microtubule-stabilizing solution pellet supernates in tubulin-stabilizing solution showed some increase in colchicine binding per tissue weight in the more dilute samples. Our data which demonstrate differences in colchicine-binding properties for total cytoplasmic and microtubule-derived pools of tubulin suggest that present assays for hepatic tubulin polymerization which assume identical binding properties should be interpreted with caution.     

Thyroid hormones and neurotubule assembly in vitro during brain development

A new model has been used to evaluate the effects of thyroid hormones on brain development. This model is based on the assumption that the major effect of thyroid hormones is in regulating the rate of neurite growth of the rat brain at early stages of postnatal development. Microtubules were chosen as markers of neurite growth. We tested, therefore, whether the rate of microtubule assembly in vitro is under thyroid hormone control. The following results were obtained: The rate of tubulin assembly into microtubules in vitro seems to be thyroid hormone dependent: (a) in 15-day-old hypothyroid rats the rates of tubulin assembly in vitro are low, comparable to those levels found in normal rats on day 3; (b) normal rates of assembly in vitro are restored upon addition of very small amounts of microtubule fragments which act as nucleating centers in the process of microtubule formation; (c) addition of microtubule-associated proteins to a hypothyroid preparation restores maximal assembly rates; similar results were obtained on adding one of the microtubule-associated proteins (purified tau protein); (d) physiological amounts of thyroid hormones completely restore normal assembly rates provided that they are administered very early after birth; (e) the ability of tubulin to assemble maximally does not seem to be permanently impaired, since normal assembly rates are spontaneously restored when hypothyroidism is maintained until an adult stage; (f) normal microtubule assembly is observed when hypothyroidism is produced at an adult stage. The model which may be constructed from these results implies that thyroid hormones are required briefly after birth to accelerate the rate of microtubule assembly thus allowing intensive neurite growth during the critical period of brain development.     

In vitro study on the alterations of brain tubulin structure and assembly affected by magnetite nanoparticles

In recent decades, considerable efforts have been made to understand the mechanism of memory, cognition, and relevant neurodegenerative diseases in the human brain. Several studies have shown the importance of microtubule proteins in the memory mechanism and memory dysfunction. Microtubules possess dynamicity, which is essential for functions of neuronal networks. Microtubule-associated proteins, i.e., tau, play vital roles in microtubule stability. On the other hand, the ferromagnetic mineral magnetite (Fe₃O₄) has been detected in the normal human brain, and elevated levels of magnetite are also observed in the brains of Alzheimer’s disease patients. Therefore, we propose that a relationship between microtubule organization in axons and brain magnetite nanoparticles is possible. In this study we found alterations of microtubule polymerization in the presence of increasing concentrations of magnetite through transmission electron microscopy images and a turbidimetry method. Structural changes of microtubule and tau protein, as an essential microtubule-associated protein for tubulin assembly, were detected via circular dichroism spectroscopy, intrinsic fluorescence, and 8-anilino-1-naphthalenesulfonic acid fluorometry. We predicted three possible binding sites on tau protein and one possible binding site on tubulin dimer for magnetite nanoparticles. Magnetite also causes the morphology of PC12 cells to change abnormally and cell viability to decrease. Finally, we suggest that magnetite changes microtubule dynamics and polymerization through two paths: (1) changing the secondary and tertiary structure of tubulin and (2) binding to either tubulin dimer or tau protein and preventing tau–tubulin interaction.     

Kinetic analysis of tubulin exchange at microtubule ends at low vinblastine concentrations

We have investigated the effects of vinblastine at micromolar concentrations and below on the dynamics of tubulin exchange at the ends of microtubule-associated-protein-rich bovine brain microtubules. The predominant behavior of these microtubules at polymer-mass steady state under the conditions examined was tubulin flux, i.e., net addition of tubulin at one end of each microtubule, operationally defined as the assembly or A end, and balanced net loss at the opposite (disassembly or D) end. No dynamic instability behavior could be detected by video-enhanced dark-field microscopy. Addition of vinblastine to the microtubules at polymer-mass steady state resulted in an initial concentration-dependent depolymerization predominantly at the A ends, until a new steady-state plateau at an elevated critical concentration was established. Microtubules ultimately attained the same stable polymer-mass plateau when vinblastine was added prior to initiation of polymerization as when the drug was added to already polymerized microtubules. Vinblastine inhibited tubulin exchange at the ends of the microtubules at polymer-mass steady state, as determined by using microtubules differentially radiolabeled at their opposite ends. Inhibition of tubulin exchange occurred at concentrations of vinblastine that had very little effect on polymer mass. Both the initial burst of incorporation that occurs in control microtubule suspensions following a pulse of labeled GTP and the relatively slower linear incorporation of label that follows the initial burst were inhibited in a concentration-dependent manner by vinblastine. Both processes were inhibited to the same extent at all vinblastine concentrations examined. If the initial burst of label incorporation represents a low degree of dynamic instability (very short excursions of growth and shortening of the microtubules at one or both ends), then vinblastine inhibits both dynamic instability and flux to similar extents. The ability of vinblastine to inhibit tubulin exchange at microtubule ends in the micromolar concentration range appeared to be mediated by the reversible binding of vinblastine to tubulin binding sites exposed at the polymer ends. Determination by dilution analysis of the effects of vinblastine on the apparent dissociation rate constants for tubulin loss at opposite microtubule ends indicated that a principal effect of vinblastine is to decrease the dissociation rate constant at A ends (i.e., it produces a kinetic cap at A ends), whereas it has no effect on the D-end dissociation rate constant.     

Direct interaction of Bcl-2 proteins with tubulin

A direct interaction between tubulin and several pro-apoptotic and anti-apoptotic members of the Bcl-2 family has been demonstrated by effects on the assembly of microtubules from pure rat brain tubulin. Bcl-2, Bid, and Bad inhibit assembly sub-stoichiometrically, whereas peptides from Bak and Bax promote tubulin polymerization at near stoichiometric concentrations. These opposite effects on microtubule assembly are mutually antagonistic. The BH3 homology domains, common to all members of the family, are involved in the interaction with tubulin but do not themselves affect polymerization. Pelleting experiments with paclitaxel-stabilized microtubules show that Bak is associated with the microtubule pellet, whereas Bid remains primarily with the unpolymerized fraction. These interactions require the presence of the anionic C-termini of alpha- and beta-tubulin as they do not occur with tubulin S in which the C-termini have been removed. While in no way ruling out other pathways, such direct associations are the simplest potential regulatory mechanism for apoptosis resulting from disturbances in microtubule or tubulin function.     

Nucleoside diphosphate kinase from brain. Purification and effect on microtubule assembly in vitro

Tubulin strictly requires GTP for its polymerization. Nevertheless, microtubule assembly can be observed in the presence of ATP as the only nucleotide triphosphate, due to the nucleoside diphosphate kinase (NDP kinase) present in microtubule preparations, and which phosphorylates the GDP into GTP. We have purified this enzyme from pig brain to homogeneity, and shown that its relative mass is close to 100 000 in its native state, and 17 000 under denaturing conditions. Therefore it is probably a hexamer, as previously shown for the enzyme from other sources, and also presents a microheterogeneity, with the major isoforms between pI 5.0 and 6.0. The enzyme is transiently phosphorylated during catalysis, as expected within a ping-pong bi-bi mechanism. The effect of the NDP kinase on pure tubulin polymerization was studied: in the presence of NDP kinase, the lag time observed in the kinetics of microtubule assembly was shorter and the final extent of assembly was unchanged. The effect of the enzyme was observed at enzyme concentrations 900-fold lower than tubulin concentration, which shows that the NDP kinase acts catalytically. Kinetic data show that the catalytic effect of the NDP kinase is faster than the rate of nucleotide exchange on tubulin under the same conditions. This result demonstrates that the tubulin-GDP complex itself is a substrate for the enzyme, which may indicate that the GDP bound to tubulin at the E site is exposed on the surface of dimeric tubulin.     

In vivo effects of zinc deficiency on calmodulin concentrations in selected rat tissues

Two groups of male Sprague-Dawley rats, one fed zinc-deficient diet, ad libitum, the other, pair-fed with the same diet, but given supplemental zinc in the drinking water (8 mg Zn++/ml) were studied. After ten weeks of diet, rats were exsanguinated and zinc and calmodulin concentrations in brain and testis were measured. Mean zinc concentration in testis was significantly decreased in rats fed zinc-deficient diet without supplemental Zn++, but mean zinc concentration in brain was not different. Similarly, mean calmodulin concentration in testis was decreased in rats fed zinc-deficient diet without supplemental Zn++ whereas mean calmodulin concentration in brain was not different. Distribution studies of zinc and calmodulin showed that both zinc and calmodulin were released more freely into soluble fractions of testis in rats fed zinc-deficient diet without supplemental Zn++. These results indicate, for the first time in in vivo studies, that zinc influences the calmodulin content of testis.