Vanadates form insoluble complexes with histones

Biochimie ( ! 997) 79. 457-402 © Soci6t6 fl'an~;aisede biochimie el biologic mol6culah'e / Elsevier. Paris

Vanadates form inso|uble complexes with histones DE Michele*, D Thomsen, LL Louters** Department of Chemistry aml Bim'hemists3; Calvin College. 3201 Burton, SE, Gramt R~q~ids. Mi 49546. USA

(Received 13 January 1997; accepted 23 February 1997)

Summary ~ Vanadium oxoanions are known to have a variety oI' physiological ellS'ors including insulin-like activity, inhibition of phosphotyrosine phosphatases, as well as direct interactions with a variety of cellular proteins such as microtubules, in this study, vanadate was found to term insoluble complexes with histories, as well as other positively charged proteins, in a concentration dependent fashion. This interaction occutTed over a 0.5-10 mM range which corresponds to the concentration range reqt,ircd for many of vanadate's known physiological effects. Results from precipitation experiments using vanadate solutions with or without the yellow-orange decavanadate indicated that the decamer form is primarily responsible for this precipitation. Vanadate was able to selectively precipitate histones from soluble chmmatin as well as from a soluble bacterial protein extract to which a low concentration ~I" histones had been added. Vanadate wa~ al:~o able to effccti,,cly precipitate histone from solutions as low as 0.006 mghnL histone. Thus, the selective precipitation of histones and other positively charged proteins by vanadate can be utilized as a tool for protein purification, in addition, this interaction may provide insight into the mechanisms for the physiological effects of vanadate. vanadate / decavanadate / histones / chromatin / insulin-mimetics Introduction Interest in the interaction of vanadate and other vanadium oxoanions with biological systems has increased steadily since vanadate was initially demonstrated to have an insulin-like activity in rat adipocytes il, 21, skeletal mt, scle 131, and hepatocytes 141. This has led to studies using v~:,nadate to investigate the mechanism of insulin action, a~ well as studies investigating the possible use of vanadium salts as an oral treatment for diabetes mellitus 15~81. The similarity of the aqueous vanadate species to the phosphate hydrolysis transition state has been the suggested mechanism evoked to explain a portion of the physiological effects ofvanadate such as the activation of a cytosolic tyrosine kinase 191, the inhibition of ion channel-ATPases[10f and the inhibition of a phosphotyrosine phosphatase i I 1!. However, in addition to its role as a phosphate hydrolysis transition state analogue, vanadate has also been found to have a variety of direct interactions with proteins such as its binding to F-actin, effects on microtubule assembly, inhibition of protein degradation, activation of ATP-sensitive-K + channels, and binding to the sarcoplasmic reticulum I 12- ! 61.

*Current address: Department el' Physiology, University of Michigan, Ann Arbor, M! 48109, USA **Correspondence and reprints

The analysis of the interactions of vanadate and cellular proteins is complicated because the composition of aqueous species of vanadate varies depending on the pH, temperature, bufl'er, and vam~date concentration 1171. Orthowmadate solutions may colttain or be converted to vanadyl (IV) IIII, to pervanad:|tes ~peroxides ~1" vanadate)1181, or to polymeric forms such as tetravanadate and decavanadatc ii 3, 1%22 I. Each of these lbrm may mediate dil'fcrent physiological effects, and therefore, assigning i~hysioh~gical ftJllction to a particular vanadate ion form is not always trivial. In studying the mechanism of the insu!inolike action of histone H4, which is known to have insulinolike active ity in adipocytes and skeletal mt|.,,cle 123, 241, we obo served that the addition of vanadate to an aqucou.~ solution of soluble histones resulted in the formation of a precipitate. In this study, this interaction is characo terized more extensively We report that several po.~itiv° ely c h a r g e d p r o t e i n s are e s s e n t i a l l y q u a n t i t a t i v e l y precipitated by 10 mM vanadate. The polymeric decavanadate is more efficient at precipitation than orthovanao date. We also report that h i g h e r c o n c e n t r a t i o n s of vanadate (25-50 raM) can selectively precipitate histories from soluble chromatin, while the DNA rernains in solution. In addition, histones are selectively precipitated by 10 mM vanadate from a bacterial protein extract to which h i s t o n e s h a d b e e n a d d e d at low c o n c e n t r a t i o n s (0.03 mg/mL). Results also indicate that 10 mM vanadatc can recover histones by precipitation from a solution as low as 0.(106 mg/mL.

458

Mate~

and methods

Mawrials t a f t thynms histones (Type ll-AS, enriched in core histones), hen egg white lysozyme, sodium orthovanadate, Hepes, poly-L-argiaine~.~l (M, 15 ~ 7 0 000), poly-L-lysine-HBr (/14.. 15 00030 000), poly-L.aspartic acid (Mr 15 000-50 000), "Iris, and mka'ococcai nuclease were purchased from Sigma (St Louis, MO, USA). Bovine ~rum albumin (Fraction V) wa~ purchased from Fisl~ Biotech (Fair Lawn. NJ, USA). l~HI-Lysine labeled histones and frozen MSB cells were a gift from V Jackson. Purified histone H4 wa~ a gift from MC McCroskey and JD Pearson of Upjohn Coml~ny (Kalamazoo, MI, USA). Purified T4 bacteriophage ly~zyme was obtained from TG Gray.

Vanadateprecipiu~thm ofpmwins I~lein solutions of calf thymus histones, lysozyme, albumin were prepared by dk~solving 1.0 mgtmL of each in KRH buffer ( 131 mM NaCk 4.8 mM KCI, 1.2 mM MgSO4, !.3 mM CaCb. 1.2 mM NaH~PO~, 25 mM Hepes, pH 7.4). A T4 lysozyme solution of about 0,5 mgtmL was obtained from TG Gray. A 0.4 M polymeric vanadate sleek (yellowoorange) was prepared by dissolving sodium orthovana~te imo the above buffer and adjusting the pH to 7,4 with dtopwi~ addition of 6 M HCI, Analysis of this stock solution using previously published methods indicates that 73% of the vanadate was in the decavanadate form 1251. The final concentrations of v:anadate ~po~ed in this pa~r represent the final concentration of van~dium ~gardless of form, ~ i p i t a l i o n ¢xl~riments were performed by adding 75 laL of protein st~k to 25 taL vanadate stocks at 22°C (final concentrations 10, !, 0,5, 1,0, 5,0, 10, and 50 raM), After mixing, samples were spun at 16 000 g Ibr 2 rain at 4~C, Pellets and sut~rnatants wet'e then analyted by 15% SDSoPAG~ and stained with C~massie brilliant blue Ro250, Percent p~tein p~¢ipitated was determined by meas~r. in8 t~ak absorbance area at t~00 nm usin~ a Gil!b~ Res~mse Series OV:Vls $~tm~tomeer (Ci~ Coming Diagno~i~sCorp, Medo i~ld; MA, USA) ~ u i ~ with a gel scanning aPl~ratus. Similar experiments we~ performed with l~)lylysine, I~)lyarginine, and I~lYasl~li¢ acid in 20 mM 'Iris buffer (pH 7,4),

Dewrmim~tion ~ vw~adaw SlY,ties ~,,vponsiblejbr precipitation A clear van~ale st~k (without decavanadate) was p~pared by ~ h k m of 0,2 M vanadate to an ~uivalent volume of I I d "Iris (pH 6,8), ~sulting in a 0, I M vanadate, 0,5 M Tris stock (pH 8,0j. A yellow-orange (decavanadate present) ~uivalent vanadate stock was pte~red by adjusting the pH of 0,2 M vanadate to pH 8,0 with 6 M HCI and then adding an equivalent volume of I M "Iris (pH 8,0), Dilutions were P r e l ~ with addition of 0,5 M Tris (pH 8,0), ~ipit~tions ~v¢~ carried (~tt as describ~ earlier substituting radiola~l~ histot~s for pmlein mixes, Bdefl~ 75 laL of 2 mghnL [,~Hl-histoncs (12~1dpm)were added:to 25 laL of vanadate stocks, The pHs tff the ~ i p i t a t i o n mixtures from e~h of the vanadate sleeks ~ t e idenlical, I%~ent protein remaining soluble was determ i ~ b y analyting SUl~matants using liquid ~intillation counting, counts were ~mrected for degradation pn~ucts by subtracting the r,~lic~a~tivitythat remained ,,aduble in 25% tdchlomacetic acid.

8fleets ~" vanadate on hi,wones bound to solubh' chromath~ MSB cells were grown in 10% fetal calf serum with medium l:! Duibecco's MEM-RPMI-1640 and supplemented with 50 mM Hepes. Exponential growth was maintained at a cell density between I x IOOtmLand 3 x 10t'linL. Nuclei were isolated with four washes of 10mM MgCI.,, 0.25 M sucrose, 0.5% Triton X-100, 10 mM Tris (pH 7,5), spinning at 1500 g for 5 rain at 4°C and collecting the pellet after each wash. Nuclei were washed once in ! .0 mM CaCI.~, 10mM MgCI.~, 10 mM Tris (pH 7.4), and resuspended in the ~me buffer at a DNA concentration of approximately I-2 mglmL (monitored by A.,t~0,DNA was digested with 50 unit,,JmL micrococcal nuclease at 37°C for ! h and digestion was terminated by addition of 20 mM EDTA (pH 6.5). The nuclei were then dialyzed against 0.2 mM EDTA (pH 8.0}, tbr 15 h at 4°C. Soluble chromatin (0,'4-1.0 mghnL DNA) was then isolated alter centril~gation at 15(11)gfi~r5 rain at 4°C, and adjusted to 20 mM "Iris (pH 7.4~, A 0.2 M vanadate stock (yellow-orange) was prepared and the pH was set to 7.4 with 6 M HCI, and then adjusted to 20 mM Tris. Dilutions were made with 20 mM "Iris (pH 7.4), in the precipitation experiments, 75 laL of soluble chromatin was added to 25 laL of vanadate stocks. Pellets were isolated as described earlier and samples were redissolved in !% SDS, Samples were analyzed using 1% agarose electrophoresis with 0. 1% SDS in TAE (40 mM Tris, pH 7.5, I mM EDTA) buffer. Gels were first stained briefly with 0, I mglmL ethidium bromide and then stained with Coomassie blue R-250. Additional samples were also analyzed with 15% SDS-PAGE to determine percent protein precipitated as described earlier.

Sele,'tive pw,'ipiuaion of histo,es.lhms a prowin extract E colt cells with an expression plasmid containing T4 lysozyme were grown and allowed to lyse. A soluble protein extract containing T4 lysozyme was obtained after removal of cell debris by centrifilgation, 211~g of calf thymus histones were added to 600 laL of the extract and this mixture was adjusted to I0 mM vanadate. A precipitale was collected and its contents as well as the contents el" the extract were analyzed by SDS-PAGE. Rccoves T ~f hismne 1t4 jhma diluW sohaions A total of 10 lag of purified histone H4 ( 10 laL of a I lag!laL stock) were diluted to final concentrations of 0.9, 0.09. 0.009, and 0,006 lagllaL histone H4, 50 mM "Iris (pH 7.4). The solutions were adjusted to 10 mM vanadate and the precipitates were isolated and subjected to SDS gel electrophoresis. The amount of protein recovered was determined by gel scanning as previously described.

Results Selective pn'cipimtion of histones by vanadate solutions in order to determine the selectivity of vanadate in precipitating proteins, a variety of soluble proteins were added to varying concentrations of vanadate as described in Materials and methods. Figure I shows percent protein precipitated at varying concentrations of vanadate with the inset indicating typical SDS-PAGE result. The minigel system was unable to separate histones H3 and H2b. These results

459 of histone precipi'ation Ibund in this experiment were very similar to the resalts seen in figure t confirming the previous experiment. Figure 2 indicates that 30c~ of radiolabded calf thymus histones precipitate at 1 mM vanadate, while 86c,; precipitate at 5 mM and 98c,4 at 25 mM vanadate. Without decavanadate, histone precipitation was markedly less over the concentrations tested. In the presence of I mM vanadate, 11% of the radiolabeled histones precipitated, at 5 mM, 21% precipitated, while 61% precipitated at 25 mM vanadate.

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Precipitation of histories from soluble chromatin

20

0' .I

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Vanadate Concentration (raM) Fig 1. Eftcots of vanadate on histone solubility. Percent protein precipitated was determined by SDS-PAGE analysis as described in MateriaL~ and methods. The vanadate concentration represents total vanadate equivalents. Data are the means ± SE from fotu" experiments. The inset shows a typical SDS gel li'om which these data are generated. Lane H is calf thymus his=ones, hines i-4 are the proteins precipitated by I0, 5.0, 1.0 and 0. I mM vanadate respeclively. Hismne H4 is indicated by open squares, his=one H3 and l12b by open circles, and his=one H2a by open triangles.

In order to gain some qualitative insight into the strength of the vanadate-histone interaction as well as to examine the effects of vanadate on his=ones in their more native physiological state, the effects of vanadate on soluble chromatin were tested. Soluble chromatin and vanadate were prepared as described in Materials and methods. Soluble chromatin (0.5 mg/rnL) was adjusted to 50 mM vanadate and the resuiting soluble and insoluble l'ra~:tions were analyzed on a i~;~ agarosc, 0.1"?~ SDS gel (results shown in fig 3). The gel was first stained with ethidium bromide (fig 3A) and then with Coomassie blue (fig 3B). This allowed us to analyze both the DNA and protein on the same gel system. Results shown in lane C indicate that 0.1% SDS effectively separates the protein and DNA of soluble chromatin resulting in

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indicate that histones H3/H2b, H2a, and !t4 showed no statistical difference in the dose dependent precipitation by vanadate. The average histone precipitation was 3% at 0.1 raM, 9% at 0.5 raM, 35% at 1.0 raM, 8()cA' at 5.()raM, 96c/~ at !0 raM, and !()0oh, at 50 ntM. Positively charged hen egg white lysozyme showed a precipitat!on paltern identical to the his=one, while bovine serum albumin and T4 lysozyme precipitated minimally, even at 50 mM vanadate (! 7% and 14% respectively) (data not shown), in addition, poly-L-lysine and poly-L arginine of similar molecular weights precipitated at vanadate concentrations equivalent to those found here ( I - 1 0 raM), while poly-L-aspartic acid remained soluble (data not shown).

Effects ol'decavana<e on h istone solubility in order to determine the vanadate species primarily responsible for the precipitation of positively charged proteins, vanadate stocks, with and without the orange-yellow decavanadate, were prepared, and protein precipitation was monitored using radiolabeled histones as described in Materials and methods. Figure 2 shows the percentage of radiolabeled his=ones that precipitated after treatment of varying concentrations of vanadate with and without the decamer tbrm. With the decamer form present, the results

8O Q.

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I

Concentratlon

10

100

of Vanadate (raM)

Fig 2. Effects of decavanadate on historic solubility. Radiolabeled histories were precipitated with vanadate stocks with and withou! the decamer form present. The precipitates were removed and the supernatants were analyzed for remaining: radioactivity as described in Mawrials and meHwds. Data .,,hown from a representative experiment are expressed as a percent of the protein that precipitates at each vanadate concentration. The vanadate concentration represents total vanadate equivalents. Squares ~.'epresent clear vanadate without decavanadate present, and triangles repre~ sent yellow-onmge vanadale with decavanadate present.

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li'ltl 3, Eft~t,~ of vaflad~tt¢on suluhl¢dar~malin, Soluble clm~m~!liu was p~l~red as tk~,~cribedin Mah,rials and method~, adjusicd to 51) mM vanadate, and uualyted by I% against, O.Iq SDS electrosL~,A, A ~ u l ~ t i v ¢ gel sL~il~,~lwith e;hidiu~ubr~!l~id¢,B, ~ n ~ ~l s~l~-xlwid~C~n~a~sieblue,La~esa~ lah¢l~l~s folk)ws: C, solut~ ¢ t ~ t i n (~)xilwately 8 lag):H, calfthymushisto~s (10 la~g I, 2, solub~ fractionsaftertl~tn~nt of 50 mM va~adat¢ih)m two experiments(~xil~ately 8 pg)~3, 4, precipitatorafter 50 mM vana° d~le l/'c~lllt~/ltOf60 and ~) lag of chmmatin,reSl~'ctively.

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an approximately 150 base pair ladder of DNA molecules ~ r l'V th)m mono-, di-, tri-, and tetranuc!eosomes, and a p~tein ~ n d which has mobility identical to calf thymus hismnes (see lane H). The data in lanes I and 2 are the soluble fractions from two separate 50 mM vanadate precipitation experiments, These results are nearly identical and indicate that this soluble fnlction,ct)nt,uns , ' " DNA, but no ~tectable protein, The data in lanes 3 and 4 are from the insoluble fractions of a 50 mM vanadate treatment of 60 and 30 pg of chromatin res~ctiv¢ly. These lanes indicate a Ct~omassie blue stained histone band, but no DNA nucleosome ladder even at higher concentration (lane 3). There is a band of ethidium staining present in the precipitated hi-

stones. This ethidium stained band is also present in the stock histones from Sigma and is likely due to the histoneSDS complex since the staining remains after DNAse I treatment (data not shown). As expected, this agarose-SDS gel system did not separate the individual histones. Therefore, similar precipitation experiments were performed using varying concentrations of vanadate and analyzed by SDS-PAGE as before. Results indicate that the average histone precipitation from soluble chromatin of histones HI, H3/H2b, H2a, and H4 was 0% at < 5 raM, 79% at 25 mM and > 96% at 50 mM vanadate (data not shown). Again no selectivity for a particular histone was apparent using these methods. The soluble lt'action from 50 mM precipitation did contain non-histone protein, but apparently at too low a concentra|ion Io be detected by the less sensitive SDS-agarose analysis.

Selective precipitation of histones./hmz a pl~tein ~:vtract In order to begin 1o assess the potential usefulness of vanadate precipitation as a protein purification tool, a soluble protein extract was spiked with a low concentration of histones (0.03 gg/~L) as described in Materials and methods. This protein mixture was then adjusted to l0 mM vanadate and the precipitated protein was collected and analyzed by SDS-PAGE. The results are shown in figure 4. As seen in lane E, the histones in this protein mixture were too diluted to be detected by Coomassie blue staining. However, when 30 times the amount of mixture in hme E was adjusted to I OmM vanadate, the hislones were effectively concentrated and were essentially the only proteins that precipitated (see lane P). !nterestingly, the T4 !ysozyme in the extract, a basic

protein, did not precipitate, Ps~cipitalio, of historic H4.from dilute .s'ul,tions The effectiveness of vanadate to concentrate histone was demonstrated by the recovery of 10 Pg of histone H4 at increasing dilution using vanadate precipitation (10 raM), The results shown in figure 5 shows that at 0.9 lag/BL, all of the protein was recovered, at 0.09 og/BL, 89% was recovered, at 0.009 BglBL, 60% was recovered, and at 0.006 BgtBL, 24% of histone H4 was recovered. Experiments were also done with solutions containing all the core histone and the results for the other histones were very similar to these reported for histone 1t4. Therel'ore, only the H4 results are reported here.

Discussion This study reports lot the first time that vanadate quantitatively precipitates positively charged proteins, such as histones, in a concentration dependent manner. Interestingly, the concentrations of vanadate required for precipitation, 0.5-10 raM, are identical to the concentrations required for

46t most of the physiological responses o f vanadatc, such as increased glucose uptake I l ~ . 26j and the installs-like activation of gene expression of certain proteins [27, 281. This study also shows that the decavanadate h~rm is primarily responsible for this precipitation. This was not unexpected, since it had been previously suggested that the binding of decavanadate to the ATP-sensitive-K + channel [151, m myosin [20[, to microtubtdes [131, and to the sarcoplasmic mtictdum [16[ involved positive charge clusters on the proteins. Thus, out" observations of the precipitation of histones, hen egg white lysozyme, poly-L-lysine, and poly-L-arginine by vanadate, support ~hese previous observations and suggest that it is likely the interaction of vanadate with basic residues that ix responsible for the precipitation. However, the observation that another basic

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.01 Concentration

.1 of

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1 (mg/mL)

Fig 5. Recovery of historic H4 Iiom dilute solutions. Puritied histone H4 (10 lag) was diluted and then each solution was adjusted to I() mM vanadate as described in MateriaLs and melhod.s. The percent of histone H4 that ,a,as recovered was determined by densitometric scaening of SDS-PAGE gels. Results are the means +__SE from lbur ex pert n l e l l l s .

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H!

H3/H2b H2a H3 Fig 4. Isolation of histones from a soluble protein extract. A soluble bacterial protein extract containing added histories was adjusted to I0 mM vanadate and the pellet isolated as described in Materials and methods. The SDS,PAGE results are shown, t,ane F is the bacterial extract and histone inixture, hme P is the precipitated proteins (fi'om 31) x more extract than in hme F), and hme H is a sanlplc of tile histories that were added to the extract. Histories arc labeled and the arrow shows location of'l"4 lysozymc.

protein, T4 lysozyme, did not prccipitzqe suggests that there may be factors other than lilt? basic residues which inlltlellcc tile decavanadatc- protein inter,'lcl ion. in addition, Ih¢ renlt~val of tile decawmadate did not completely eliminate prc o, cipitation by vanadale. This may indicate that other species may bc capable of similar intcraction,s ~tt higher concentrations. Fbr example, tetrameric vanadate may be capable of such ino teractions. It is reported thai telrameri¢ vasari;lie binds basic residues in superoxkle dismutase and myosin 119, 201. The effects of vanadate on the mlubility of histtmes in their more native physiological state (in chromatin) were also examined. Although slightly higher concentrations of vanadate were required to precipitate the histones, at 50 mM vanadate virtually all the histories precipitated while the DNA remained soluble, This suggests that the vanadate°hi° stone interaction can successfully compete with the DNAhistone interaction in chmmatin and is capable of disrupting nucleosonlc structure. This study also demonstrates that vanadate selectively precipitates histones Ii'om a soluble protein extract and is able to concentrate histones from dilute solutions. Taken together, these observations suggest the possible efficacy of using vanadate precipitation in protein or DNA purification procedures. The histone precipitation by vanadatc reported in this study likely results from the combined effect of positive

~2 charge neutralization and protein cross-linking by vanadate anions, There are several arguments that strongly suggest that this pr~zcipitation requires a direct interaction of vanadale and protein and that it is not simply a salting out phes o l , non, First, other anions, such as phosphate and sulfate, ...... these low concentraisolated ~llets were p~cipitates with the his(ones, This was es~cially evident in the lower concentration vanadate solutions where the supernatants obviously c l ~ f i e d as the decavanadate and protein precipitated. And finally, t ~ precipitation of the histones, but not the DNA, from ~luble chmmatin suggests a direct his(one vanadate interaction. Finally. the possible physiological relevance of these resuits should not ~ ignored. The data reported here suggest that a ~rtion of the physiological ctle~;t, "" " s of vanadate may attributed to a direct histone-vanadate interaction. There have ~ e n reports of vanadate having effects on gene expression of particular genes 127, 281. Vanadate is also known to result in increased DNA synthesis and has mitogenie effects [29, 301. Metavanadate is also known to induce mitotic gene conversion and reverse point mutations in yeast [3 II. Although these effects have been attributed to the interaction of vanadate with cytosolic factors, including the insulin pathway and microtubule assembly, the possibility still exists that a vanadate-histone interaction is at least partly responsible for these physiulogica! et't~cts. !n conclusion, it is clear from this study, that v,anadat~, interacts very strongly with positively charged proteins, including histones, and understanding the direct interactions of vanadate with cellular proteins and enzymes may he an hnport-ant key to understanding the physiological ftltl¢tioit,s of Vll n ~1d i tl Ill 0 X oa n i 0 n ~,

Acknowl~gmen~ This iv,seai~h was funded by Bristol Myers Squib Company Award fl~m~Research Corporation and Howard Hughes Medical Institute Ui~rgraduate Research Fellowship, We thank Vaughn Jackson for his gift of [aHi-la~led his(ones and frozen MSG cells, We thank Mark McC~)skgv and Jim Pearson for theircontributionof purified histol~ HI and Terry Gray l't~rpurified T4 lysozynle, Referents I Dubyak GR, Kleiatellcr A ~1980~ The insulin-mimetic etlL'ds el" ~aa adale in isolated ral udil~ytes, ,I bl6d Chem 255, 5311~- 5312 2 Sttc~hler Y, Karlish SJD ( 198111 Insulin-like xtimuh~titm uf gh.tcmc o,~idalioll il~ral adil~aeyt~s by ~'andyl (IYl i~ms, Nl~lt,'e 284, 556-558 3 (?lark AS, FaganJM, Milch WE 11985} Selecti,,,ity of the iasuln-likc action of vanadate on glucose and pr~leiil metabulislu iu skeletal mu~le, ii~whem J 232, 273=276 4 Jackson TK, Salhaaick AI, Sl~wks ,ID, Sparkx CE, ll~dugaiao M, Asiatruda JM 119881 Insttlh~-alit~lic etlkxts ot" vanadate in prhaary cultui~s of rat h~patt~ytes, Ditd~.lr,~ 37, 123: ~24!)

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