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Isoelectric acid focusing degrading of indoleacetic enzymes from pea roots. C.A. van Botanisch der Mast Utrecht Laboratorium, SUMMARY In homogenates IAA. It protein neutral as at complex least of 3 other commonly displayed by two an possesses At least pH. only of pea roots consists one which isoelectric peroxidases the is protein complex enzymes of point are 9.5 and with present only similar a the intimately has degradingprotein. Probably IAA for responsible probably are net small a This charge of molecular range these degradation of associated. at weight enzymes are also bound to membranes. 1. INTRODUCTION Several studies IAA-oxidase from on Sequeira & gel chromatography. distinct fractions Cronenberger Janssen roots. one of capable al. et (1969) found found same can with small niques offer only one enzyme group dilferences in The two roots. with pea of peas in which indicating different types of enzyme molecules One type is roots. the presence can Use be showed of gel that the tech- specific to a another column of presence IAA in degrading chromato- compounds of other assigned indicating enzyme But resolution of inadequate. results capable peroxidative a is activity. the of IAA already gives of peak because weight (1969) support, i.e. polyvinylpyrrolidone, of pea one degradation author present to separation molecular is necessary before the enzyme. presence working in roots other, possibly of of tobacco fraction when one with performed enzyme in both fractions. Most authors ascribe graphy the to the homogenates enzyme fractions two fraction could be converted of the only have been plants (1966) reported 1AA in degrading (1966) of roots Mineo two homogenates while the other type is non- peroxidative. Another difficulty IAA-oxidase peroxidase causal as In the activity electric is present pH point Acta Bot. Neerl. ubiquitous is difficult presence of study use gradient is is reached. June to has et a peak prove with are on of IAA 1967). The various a peak degradation. and But proof of a is assayed. been made of isoelectric al. Identification of the fact that gel chromatography Under established in which 1970 peroxidases. commonly founded homogenates (Vesterberg 19(3), is coincidental with when crude electrophoresis a is the peroxidase relationship impossible current a proteins peroxidases focusing, influence are of move easily a the form of applied until their iso- resolved by this 363 C. when technique column of Sephadex MATERIAL AND Seeds of Pisum earlier Me 10 MgCl2 . instructions in the vessel stepped from The buffer out of compound 7.5%. Eight gels ml duration of tained at gradient was 3/4 as some were 4 ml The focusing The applying in 15 the this of 1 cm gels an were but ml these In disturban- The column obtained. were addition of this first into method 30 during gel the experiments omitted later the cm higher was were the at 20% and potential 25 cm were was a main- pH cathode measured 406 M 5, in nr glass sucrose. of the The V/cm. membrane. and immersed in 1 ml 50 ppm IAA and 2 ppm in or sucrose was gel. Drifting resulting pH’s of 1 3 used from the electrode solu- is level with the pieces as influence no acrylamide carrier which was excess minutes as Ampholine carrier, 40% overnight residual IAA the to of the Ingold microelectrode, extension, out the diameter of 0.5 the a run an cut avoid according and 2.6 mA per potential, with mA. gradient. low, separated containing V/cm carried and the the and buffer, pH 5.0, containing was a 16-17 hours in 12 bridge, through incubation is current was and cm gels water 1 polymerisation In the This step containing on was at instances the then removed cold determined room. the by p-coumaric The pieces addition of Salkowski’s reagent. Gel 364 of distilled intervals of Me Ilvaine’s acid. of 300 V, in the at to 1/4 to maintained pH-ranges used, kept to Ampho- in the different fractions. performed was the lower electrode. After at which the salt In ml layered negligible was gels was solution pH persulphate. length V, resulting 250 taken in the a Usually 3/4 simultaneously. and the enzymes of the of 2-2 the added was was all at was steadily region to described earlier in Me Ilvaine’s buffer. (1968). by hours case in Stockholm-Bromma, detectable. The concentration of was with 20-24 out contained according temperature Fractions as focusing Fawcett removed was which 7.4, upper electrode carrier Mitchell (1967). by The in the acid pump. of the with ammonium persulphate by and carried was 27000 g supernatant decreased the as tested was was current taken was isoelectric carried The a been described as grown performed was in the former eliminated variations of (1968) recommended of the focusing the V; of IAA were (LKB-produkter, solution. voltage peristaltic a Polyacrylamide of Wrigley tion enzymes roots buffer, pH focusing 4.5 ml lighter buffering capacity this have volume of 110 ml. The concentration of the coagulating compounds virtually of the the the kathode degradation the As a 700 to up drained with was isoelectric %. Usually containing experiments ces 1 Tris-HCI manufacturer 3-10 and 7-10. The pH latter the in or The duration of the 2°C. at i. e. pH 5.0, column with was of the 1969). Homogenation Liquid from a line carrier degrading Gele Krombek” “Vlijmsche cv. Mast Ilvaine’s buffer, Sweden) containing Fractions obtained from gel. IAA MAST METHODS sativum der (van mM G-100, polyacrylamide VAN DER this method. analyzed by 2. in performed A. chromatography on Sephadex G-100 was performed in Acta Bot. Neerl. columns with 19(3), June 1970 ISOELECTRIC FOCUSING dimensions of 35 OF lAA DEGRADING ENZYMES 1.9 X tions after the passage of destilled focused polyacrylamide on with mentioned. out on retained strength proteins 35 were effected was The isolation of membrane-bound IAA in a The model L 50 Spinco pellet teflon resuspended was C-25, 1.9 with degrading with the aid of a up 1 ml and acidic cation elution strength was 0.06. buffer same in sucrose weakly The ionic the 40% perThe by raising obtained. 48000 RPM at a cm. and were and enzymes during glass cylinder 1 carried was hour and a in out 50. rotor tightly fitting piston. Peroxidases Total Dubin 3. centrifuge 6.5 pH 0.3. Fractions of 5 ml to carrier X taken lyophilized, CM-Sephadex buffer, NaH 2 P0 4-Na0H removal of the the ionic as gels carried was Ampholine obtained. The first 8 frac- were were the The column dimensions exchanger. formed containing water exchange Ion Fractions of 5 ml cm. of the void volume were located with benzidine phosphate determined was reducing as according to agent. the method of Ames & (1960). RESULTS isoelectric Liquid rier with One Fig. 1. a range peak Liquid carrier anode the pH of IAA isoelectric of pea 3-10 focusing a root gives degrading establishing of gradient figure. Peroxidases Bot. Need. are activity pea of found IAA is localised 19(3), June 1970 supernatant using the results root pH solution. The last 6 fractions gradation of Acta focusing of is represented apparent with supernatant during 3-10 was consisting used. anl.E.P. Ampholine an isoelectric 20 hours. An The first 6 fractions of the cathode in all the fractions around an between car- in fig. I. solution pH 3 are and point Ampholine contain the not shown in pH 11. The de- of 9.5. 365 A. C. 2. Fig. isoelectric Liquid size foregoing figure. of is the 9.5, one broad. same as one but the broadness mask of such was of 8.4 of is this Nearly ding all an were enzymes showed IAA of these the enzymes pea with 366 Sephadex on of 5.6. was performed broader, than the of 20 fraction gradual I.E.P. an of presence only in in a in at pH fig. only was a der the Mast the ones of only pH in of 8.4. 1970) and one Peroxidase containing 3-10 pH due a to at root a pH by 9.5 gradient 9.7 were subsequently distribution of enzyme Acta pro- activity, the electrode reduced was degrading to ribosomes peak focused pea pH 7-10, only of IAA. probably gra- slope of gradient 5.6 all the fractions between this and the activity are pH to was present. 1.2 and 6.5. When IAA pH membranes and pH If I. region coagulated compounds This is a fractions with of IAA peak with enzymes 3-10 and the a gradient is rather peak although given one pH degradation second a In this supernatant (van the was become not situated between activity G-100, around this and degrapH 9.5 continuous release from the membranes and the concomitant G-25 Sephadex indicating MAST during 7-10 even more degrading and refocused in focusing 20 hours and all the ed is gradient is still localised IAA focusing gradient a for the hours, Removal of root pH gradient. The pH gradient in all fractions outside the found abolished this When in degrading activity. kathode. totally I.E.P. phosphate of the of the could be found. This all indicates that responsible instead of 20 found with of the of other gradual more solutions. If the duration of 10 hours did pooled activity apparent was presence peak even were slope does not increase If, however, first focused 9.7 to peak complex however, is gradient a supernatant same {fig. 2) peak gradual slope the different I.E.P. ’s. dient of 7-10 tein the to of the The side degradation of IAA 1. The of gradient a acid DER for IAA conversion. proteinresponsible This could slightly pH in the depicted in fig. of 9.5. Due (I.E.P.) the supernatant in been omitted at the have than the a of pea root focusing hours. Several fractions VAN migration towards ultracentrifugation was of then apparent. 3-10 pH pooled, during concentrated gel-chromatograph- molecules Bot. Neerl. was the 19(3), same June 1970 ISOELECTRIC Fig. 3. Ion FOCUSING OF lAA exchange study 0.06 strength with membrane-bound tein molecules. strength. No DEGRADING of IAA counterions. as while enzymes, The at degrading enzymes sodium elution influence ENZYMES the retained of this retained of the flow rate on pH 6.5 on The first peak peak CM-Sephadex of peak is activity consists of free IAA can be the retention effected was C-25 at ionic largely to the ionic by raising detected due degrading pro- although this pa- 2 rameter in the as various experiments found after addition of KC1 present in the form can Ion high peaks of the void organelles because of on a consists but only /hour. i.e. 1969), no enzyme is its lower molecular degrading removal IAA degrading activity in activity of these of these this cell not to molecules from this first peak. retention of all IAA the column. Ion due is peak most at this weight counterions way of retained orcinol is activity possibly Further the degrading lowering elution with enzymes, even strength 0.06 did not the ionic destilled the column. by Most of the enzymes due the to relatively strength of the free to a of the enzyme strength by carrying water resulted membrane-bound rise as The disappeared. in retained total molecules, by of isolated membranes with give These buffer, eluant test. the ionic 0.005 resulted in total removal and the membrane-bound to of this rise gives elutes after the passage pH not pH. Lowering exchange chromatography tion buffer of ionic 6.5, confirmed with the removed in this elution buffer from 0.06 the therefore are structures molecules homogenation pH at as peak of membranes and ribosomes. mostly and was 3. The first fig. charge I.E.P., of ribosomes IAA the Mast der (van ml/cm C-25 with sodium shown in negative of their lower charge as volume and after out urea weight peak CM-Sephadex activity have The presence weak or between 8-25 then be detected. exchange to two molecular varied peak an of eluIAA degrading activity. Acta Bot. Need. 19(3), June 1970 367 C. Fig. 4. Polyacrylamide isoelectric focusing tion with were adjusted, fraction 3 of 9.3 pH a after portions only Separation the small with The in each the IAA present. gel. Due were dient of Due Pea two I.E.P. itself in this always present 16 pH, time. It in the pH net hours associated fraction of pH a 6 3 and of IAA charges of this protein 5 have reached occur. The around enzymes to the enzyme band elute from the of can be inferred from the can first focused was in liquid given seen faster fraction of 9.0 similar results in several although fig. moving pH a following of gradient to took pH 7,6 place in and a 5, gra- in fig. 4. from 9.3. were raising isolated and divided into 3 portions. 7.5% polyacrylamide enzymes in be con- indicate colour achieved column after of the enzyme molecules in the 3 focusing with the initial or are charge the I.E.P. 0.3. types was of 9.3 3% on their acrylamide denotes that the blue hatching faster in the various pH areas In this to the does not to portions 5, respectively. is confined MAST The frac- The portions. with citric acid after addition of the carrier varying of the after net DER degrading enzymes. pH 7, adjusted stippled the enzyme 7-10. The results adjustment to VAN to red. supernatant Refocusing the enzymes The %. degrading root adjusted pH to 7.5 IAA divided into degradation varying of the elution buffer 3-10. The fraction with These and carrier, The to the degrading turns quickly IAA strength respectively. 368 are was The association of the pH liquid focused isolated addition of the amount of diffusion while benzidine experiments: of was of the two types of molecules enzyme employed retained the ionic the peroxidases moving protein centration (see fig. I) A. gels the have pH 4 that protein. portions has degradation Two other established of IAA peroxidases experiment the but in the with fractions with Acta Bot. after yet reached their gradient the Repeating found, not Neerl. 19(3), pH June is are the 9.5 1970 OF ISOELECTRIC FOCUSING Fig. 5. lAA DEGRADING isoelectric Polyacrylamide focusing gelchromatographed on Sephadex addition dases of the possessing concentration the ± carrier, pH 0.1. of 9.4 gradient. AH the except branes as positive charge 7.5 %. The degradation of at this of the 8 this pH pH fractions of the pH pH at the with fraction peroxidases degrading protein. The the various fractions is indicated are same the 3th 6 8 cm in the of the due to the the large same benzidine the gels an amount of was I.E.P. mem- range of molecular same protein in shading. Hatching to red turns measuring around of the differences in acrylamide for various gels The only peroxi- The used 5.0. was that figure. relative concentration achieved with buffer was point present in by corresponding colour in supernatant root pea implying, fraction to of elution to 6.5, shown are IAA is localised in the gel corresponding indicates areas where the blue first The gel corresponding here. Three other the IAA of the G-100. pH 7-10, raises range The variation in the present weight a was ENZYMES within a few minutes. and 9.7 the slowest moving protein 3 migration the % acrylamide In the next on polyacrylamide with Only peroxidases ones being displaced peroxidase when the absent. In pea root with gels supernatant was concentration of a degrading protein was twice with gels corresponding Acta Bot. Neerl. an from the cut into fraction 6 19(3), June freeze-drying. positive charge gel I.E.P. of 9.4. were to after a 1970 at The results pH 6.5 are are shown in shown, the towards the anode. All the fractions Only pieces this protein showed and incubated with showed IAA fast. as gelchromatographed G-100. The first 8 fractions after the passage of the void volume Sephadex focused experiments was of the IAA fig. 5. negative display degradation IAA. on were a of IAA However, the gel degradation throughout the gel till 369 A C. the 13th These transition deviating between result in is proteins this the the solution and degrading the membranes in until anode may then enzymes this reach the they electrode I.E.P. This protein dative at fig. solution. The account for the but which all one to an tive protein to the the other was possible, and of the (Margolis & travelled twice at a not a Mast as fig. is as It compound. was and of apparently non-peroxi- a showed the presence fraction assumed that this also were proved now located because in the was was non- after removal of all of peroxidase capable a fast least have the separation in degrading IAA, as be of separately move should then be degrading enzymes sodium ions. an equal size is present. At the IAA At is gel a 50 was and indicating found at the 3% two the of way. 7.5% 150 interference outside still happen nm band enzyme of the separation never same concentration of nm latter concentration But It amount which should polyacrylamide gels, were performed with was distributed over each molecule in the in the and nega- positive exchanger and elution 7.5% polyacrylamide, the by did types peroxidase the presence of non-peroxidative IAA degrading band. seen was ratio of ion associated intimately are concentrations of 7.5% acrylamide. that in all the fractions present with evidence on an strength increasing 1967). as same when all the IAA ionic band, indicating can enzyme molecules pore diameter of this degradation is of difference in Kenrick 5 it protein 1970) of IAA peroxidative that both types of enzymes have not occur mean and the free IAA 370 at zero of molecules in this From two I.E.P.’s possessing degradation a peroxidases case, even at course, did enzyme enzyme IAA the concentration of occur positive of small acrylamide gel types negative charges separation a not gradient positive only ones far-fetched. When both enzyme types should they exchanger very shallow when present. But are In that paper another enzyme this to assumption seems not remains Also complex remain valid. The to polyvinylpyrrolidone on This indicates that This possible. promotes the there still remains I.E.P. of 9.5 bound peroxidases the one 4. fig. charges. only But in fig. 4 is shown that seems identical with 1969). adsorb not This peak. two more this conclusion enzyme molecules, i.e. The conclusion that both to protein of IAA, 5. Mast der peroxidases shown in IAA degradation chromatography (van peroxidase, other in two mentioned that did adsorbed of this complex least as one, of the latter for the probably are depicted 1 and 2 it could be apparent that figs. degrade peroxidases consists of in given not 9.0 and 8.3 a carrier of the presence towards MAST gel. responsible around these do other a the the to move DER DISCUSSION even as will release of bound IAA From the results of due probably structures zone subsequent 4. This is cm. fraction. VAN an I.E.P. presented degrading enzyme of 9.4. indicating In a displaying degradation previous that both paper (van of der the membrane-bound molecules consist of the Acta Bot. two Need. enzyme 19(3), types June 1970 ISOELECTRIC lAA FOCUSING OF mentioned. The DEGRADING range of molecular large found after gel enzymes continuous release of chromatography indications for It can of range larger the as degrading weights. IAA This three nevertheless by in this the cover also attached are peroxidases a factor peroxidases stimu- 2-3 shows by gel a to not ca- they as process with protein identical a show the these molecules peroxidases specific to during found in are of discrimination between these groups of enzymes possibility due state be communicated later. ribosome-bound of several overlap free phenomenon participate the degrading other, basic, peroxidases these or IAA probably the to degrading protein, i.e. Possibly of IAA degradation communicated). IAA Membrane-bound structures. this from fig. 5 that seen molecular of pable late also be phenomenon this free is Sephadex Further evidence will electro-focusing experiments. same of the weight on membrane-bound molecules Several chromatography. ENZYMES be (to the im- chromato- graphy. Due the fact that the IAA to lose Galston was in or due starch, from peroxidases to intact cellular dases found in this material to other anodic cell ments A dases achieved of the two positive It is material IAA the in case anodic to gel This is due ion exchange differences in the charge to open that peroxi- membranes or from peas. the to restricted peroxi- diffusion structure. and electrophoresis, Mast question, the 1969) probably of these enzymes. The enzyme molecules non-peroxidative phenols, although der & present was electro-focusing experi- degrading protein gel. cellu- Siegel They presumed that the possible on I and fig. 3 shows the better resolution of the polyvinylpyrrolidone (van by indications. faulty phases, two performed types of enzymes composing the IAA degrading protein inseparable by of the when in fact cathodic but attached polyacrylamide protein imposed by As the be fig. as of Pisum sativum. roots This is also structures. between on introduce can organelles. are of short duration for the comparison such present in are enzymes duration, noted that acetocarmine (1967) already anodic among this strips acetate degrading of short electrophoresis experiments still to seems separation can to on be accounted for not that assumption this seem achieved is compound adsorption due to poly- valid. ACKNOWLEDGEMENT gratefully acknowledges The author the technical assistance of Miss M. A. T. Gebbink. REFERENCES Ames, B. N. & D. T. Dubin phage deoxyribonucleic Cronenberger, L., ses” des Fawcett, letters Janssen, racines J. S. (1960): R. Ville de Pisum (1968): The role of acid. J. Biot. & H. Pacheco sativum. Isoelectric polyamines Chem. Bull. (1966): Soc. fractionation in the neutralization of bacterio- 235: 769-775. Purification partielle des “auxine-oxyda- Chim. Biot. 48: 833-836. of proteins on polyacrylamide gels. FEBS 1: 81-82. M. G. H. (1969); The form of indoleacetic acid oxidase of pea roots. Acta Bot. Need. 18: 429-433. Acta Bot. Neerl. 19(3), June 1970 371 C. A. VAN DER MAST Margolis, sieve Mast, of — J. & K. G. Kenrick Nature gradient. C. A. van der (1970): Separation Mitchell, gels. W. M. Sequeira, L. (1967); Biophys. & L. Mineo from tobacco Siegel, Bot. roots. A degrading Neerl. IAA from pea roots oncolumns 620-626. enzymes source Partial Physiol. B. Z. & A. W. Galston degrading enzymes 18: from pea roots on columns of polyvinyl- of electrophoretic artifacts in polyacrylamide 171-174. 147: (1966); Plant molecular 141-146. 19: potential Acta across a 1334-1336. Acta Bot. Neerl. of IAA II. Acta Biochim. 214: (1969): Separation of polyvinylpyrrolidone. pyrrolidone. (1967): Polyacrylamide gel-electrophoresis (London) purification 41: The (1967): and kinetics of indoleacetic acid oxidase 1200-1208. isoperoxidases of Pisum sativum. Plant Physiol. 42: 221-226. Vesterberg, Studies O., on terization T. Wadström, extracellular of enzymes K. proteins and Vesterberg, from toxins isoelectric by H. Svensson Staphylococcus aureus. focusing. & I. B. Malmgren Separation Biochem. (1967): and charac- Biophys. Acta 133: 435-445. Wrioley, by 372 C. (1968): isoelectric Gel electrofocusing focusing. Science Tools - A 15: technique for analyzing multiple protein samples 17-23. Acta Bot. Neerl. 19(3), June 1970