Physarum polymalic acid hydrolase: Recombinant expression and enzyme activation

Biochemical and Biophysical Research Communications 377 (2008) 735–740

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Biochemical and Biophysical Research Communications j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y b b r c

Physarum polymalic acid hydrolase: Recombinant expression and enzyme activation Wolfgang Mueller, Markus Haindl, Eggehard Holler * Bio­phys­ik und Phys­i­ka­li­sche Bi­oche­mie, Uni­ver­si­taet Re­gens­burg, 93040 Re­gens­burg, Ger­many

a r t i c l e

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Article history: Received 12 September 2008 Available online 7 October 2008  Key­words: Recombinant expres­sion in Esch­e­richia coli and yeast Gly­co­syl­a­tion Zymo­gen Hydro­lase acti­va­tion by phos­phor­y­la­tion Plasma mem­brane pro­tein tyro­sine kinase Secre­tion Mem­brane dam­age Plas­mo­dium phys­i­ol­ogy Bio­tech­nol­ogy Na­no­con­ju­gates

a b s t r a c t As a plat­form for syn­the­ses of na­no­con­ju­gates in anti­tu­mor drug deliv­ery, polyma­lic acid together with its tai­lor­ing spe­cific exo­hy­dro­lase is puri­fied from plas­mo­dium cul­tures of the slime mold Physa­rum poly­ ceph­alum, a mem­ber of the phy­lum myx­omy­cota. Polyma­lic acid hydro­lase is expressed in an inac­tive form that func­tions as a molec­ul­ ar adapter for polyma­lic acid traf­fi ck­ing within the plas­mo­dium and is acti­vated only dur­ing secre­tion. Acti­va­tion fol­lows spe­cific pro­tein tyro­sine phos­phor­y­la­tion and dis­so­ ci­a­tion from plasma mem­branes. Puri­fied inac­tive Physa­rum polyma­lic acid hydro­lase, recom­bi­nantly expressed in yeast Sac­cha­ro­my­ces, is acti­vated on a preparative basis by the addi­tion of plasma mem­brane frag­ments from plas­mo­dia of P. poly­ceph­alum. Acti­va­tion of polyma­lic acid hydro­lase and inhi­bi­tion of polyma­lic acid syn­the­sis by pro­tein tyro­sine phos­phor­y­la­tion are com­ple­men­tary events and could indi­ cate a joint sig­nal response to plasma mem­brane dam­age. © 2008 Else­vier Inc. All rights reserved.

The nat­u­ral sub­strate of polyma­lic acid hydro­lase (PMase) is polyma­lic acid [b-poly(l-malic acid), PMLA] [1]. The unique poly­ es­ter receives high inter­est as a molec­u­lar car­rier of espe­cially nucleic acid inter­act­ing pro­teins in the mul­ti­nu­cle­ated giant plas­ mo­dium of acel­lu­lar slime molds (my­xo­my­co­tae) [2–5] [and ref­er­ ences therein] and, recently, in nano­med­i­cine as a bio­de­grad­able, non­toxic, non­im­mu­no­gen­ic poly­func­tion­al plat­form for the syn­ the­sis of drug deliv­ery na­no­con­ju­gates called Po­ly­ce­fins [6,7 and references therein]. PMLA is highly expressed in the plas­mo­dium of Physa­rum poly­ ceph­alum [8] to aid the dis­tri­bu­tion of nuclear pro­teins which are involved in syn­chro­nous nuclei divi­sion and cell growth [9,10]. PMLA and PMase are jointly expressed [1,4,11], and the pro­tein, not enzy­mat­ic­ ally active in the cell, has been attrib­uted the func­tion of a molec­ul­ ar chap­er­one of PMLA [9,12]. PMLA is con­sti­tu­tively expressed [8] and engaged in mega pro­tein com­plexes in nuclei and cyto­plasm [9]. Sur­plus PMLA and the acti­vated PMase are secreted and can be har­vested and puri­fied from the cul­ture broth [1,12,13]. The enzyme has been char­ac­ter­ized in detail and found to be a valu­able tool for bio­tech­ni­cal tai­lor­ing of PMLA [1,14]. How­ ever, puri­fi­ca­tion of the pro­tein is extremely trou­ble­some and does Abbre­vi­a­tions: PMse, poly­mal­a­tase, polyma­lic acid hydro­lase; PMLA, polyma­lic acid; ATCC, Amer­i­can type cul­ture col­lec­tion. * Cor­re­spond­ing author. Fax: +49 941 943 2813. E-mail address: egge­hard.hol­ler@bi­ol­o­gie.uni-re­gens­burg.de (E. Holler). 0006-291X/$ - see front matter © 2008 Else­vier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.127

not allow pro­vi­sion of large amounts of pure enzyme. The mode of acti­va­tion is unsolved, although acti­va­tion by lim­ited cleav­age of a high molec­u­lar mass zymo­gen dur­ing secre­tion has been sug­ gested [12]. In the pres­ent com­mu­ni­ca­tion, a method for the con­ trolled acti­va­tion of PMase and a route for recombinant syn­the­sis and puri­fi­ca­tion will be pre­sented. Mate­ri­als and meth­ods Pro­te­ase inhib­i­tor cock­tail was bought from Roche (Basel, Swit­zer­land). Tyro­sine-spe­cific pro­tein-phos­pha­tase (EC 3.1.3.48) and Tyr­pho­stin-47 were obtained from Cal­bio­chem (San Diego, CA) and enzymes for recombinant work from Invit­ro­gen (Kar­lsruhe, Ger­many), Promega (Mad­i­son, WI, USA), Roche Diag­nos­tics (Mann­heim, Ger­many), New England Bio­labs (Bad Sch­wal­bac, Ger­many), and kits from Ma­chery-Na­gel (Du­er­en, Ger­many), Qui­agen (Hil­den, Ger­many), Promega (Mann­heim, Ger­many). All other chem­ic­ als were pur­chased from Merck (Darms­tadt, Ger­many). Anti-PMase anti­body against key­ hole lim­pet hemo­cy­a­nin (KLH)-con­ju­gated oli­go­pep­tide NH2CA(203)GGVEHPVYPEGKWR (217)-CONH2 was cus­tom made and affin­ity puri­fied by Pin­e­da (Ber­lin, Ger­many). Anti-His6-per­ox­ i­dase was from Roche Diag­nos­tics (Mann­heim, Ger­many) and anti-rab­bit IgG-per­ox­id ­ ase (2nd anti­body) from Pierce (Rock­ford, USA). Prim­ers for cDNA clon­ing were 59Nhel, 59-TAA TTA CAT GAT

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GCG GCC CTC TAG-39 and 39Notl 59-ATA AGA STG CGG CCG CTA GTT TGC TCA TTT CTT GG-39 and for cDNA with­out sig­nal pep­tide 59Hin­dIII_SP, 59-AGC TAA GCT TAA AAA TGG GAA GTG GCC CGG AAC AAA CGT TCT TC-39 and 39Str­ebb 59-ACG TGA ATT CTC ATT TTT CGA ACT GCG GGT GGC TCC AAG CGC TGT G-39. Plas­mo­dia of P. poly­ceph­alum yel­low, wild type, high PMLA pro­ ducer M3CVII (ATCC 204388) were grown as described [13]. For clon­ ing and recombinant expres­sion of PMAse, the Esch­e­richia coli strains NEB 5a, NEB T7 RP and NEB T7 Rosetta (New England Bio­labs, Ips­ wich, MA, USA) were used and for expres­sion in yeast Sac­cha­ro­my­ces ce­re­vi­si­ae strains c13-ABYS-86 [15] and S. ce­re­vi­si­ae W303-1A [16]. mRNA iso­la­tion, RT-PCR and DNA-clon­ing. The gene of P. poly­ceph­ alum PMase com­prises 2233 bp and includes a 42 bp 59-untrans­lated region, a hydro­pho­bic leader pep­tide MHSRSACIFVLCGLLPFVLG, the coded pro­tein sequence, and a 71 bp 39-untrans­lated region with a short oligo(A) tail (Gene Bank Acces­sion No. AJ543320). Total RNA of 2 days old Physa­rum plas­mo­dia was iso­lated with the Rneasy kit (Qui­agen, Hil­den, Ger­many) and ana­lyzed by aga­rose (1%) gel elec­tro­pho­re­sis. Tran­scrip­tion into cDNA was car­ried out with Super­Script™ III Reverse Trans­crip­tase (Invit­ro­gen, Kar­lsruhe, Ger­many) and Oligo dT18 primer. The PMAse cod­ing sequence was ampli­fied by stan­dard PCR-meth­ods using Phu­sion™ High Fidel­ ity Poly­mer­ase (New England Bio­labs, Ips­wich, MA, USA) and gene spe­cific prim­ers 59Nhel and 39Notl. For expres­sion in E. coli, PCR frag­ments puri­fied by aga­rose gel elec­tro­pho­re­sis were digested with NheI/NotI and cloned into the pET24a(+) vec­tor (Invit­ro­gen) in-frame with the C-ter­mi­nal His6tag. For expres­sion in S. ce­re­vi­si­ae, sequences were inserted via HindIII and EcoRI in the pYES2 shut­tle vec­tor includ­ing N-ter­mi­ nal Ko­zak trans­la­tion ini­ti­at­ ion sequence and C-ter­mi­nal His10-tag. After T4 DNA-Ligase liga­tion (Fer­men­tas, St. Leon-Rot, Ger­many), E. coli NEB 5a (New England Bio­labs) was trans­formed and con­structs were ver­i­fied by DNA sequenc­ing at Gene­Art AG (Re­gens­burg, Ger­ many). S. ce­re­vi­si­ae strains W303 and c13 were trans­formed by the LiAc method [17]. Plas­mid DNA from E. coli was iso­lated with QIA­ prep Spin Mini­prep kit (Qui­agen) and DNA frag­ments after aga­rose gel elec­tro­pho­re­sis with QIA­quick Gel Extrac­tion kit (Qui­agen). cDNA devoid of sig­nal pep­tide, was PCR ampli­fied with prim­ers 59Hin­dIII_SP and 39Str­ebb and cloned into vec­tor pYES2. Recombinant expres­sion. E. coli NEB T7 RP and NEB T7 Rosetta (New England Bio­labs) grown at 37 °C in LB-media. Pro­tein expres­ sion in 5 L was induced with 0.1 mM IPTG at an OD600 of 0.6 After 12 h at 15 °C, pel­leted cells (3000g for 10 min) were resus­pended in extrac­tion buffer (Tris–HCl 100 mM, pH 7.5, KCl 300 mM, PMSF 1 mM, imid­az­ole 10 mM) and son­i­cated (Bran­son son­i­fier, He­ine­mann, Ham­burg, Ger­many). After cen­tri­fu­ga­tion at 20,000g for 20 min at 4 °C, sol­u­ble cell extract was sub­jected to Ni-che­late chro­ma­tog­ra­phy. Sac­car­omy­ces ce­re­vi­si­ae W303 or c13 was grown at 30 °C in SD-medium with­out ura­cil con­tain­ing 2% glu­cose until an OD600 of 0.8. Cells pel­leted at 3000g for 10 min were sus­pended in 5 L of induc­tion medium con­tain­ing 2% gal­act­ose. After 12 h at 30 °C, pel­leted cells were sus­pended in extrac­tion buffer and lysed with glass beads equipped dis­rupter (Frit­sch, Idar-Ober­stein, Ger­many). Cell frag­ments and nuclei were pel­leted at 3000g for 15 min at 4 °C before cell mem­branes were sed­i­mented at 20,000g for 20 min. Pellet after 2 h treat­ment at 4 °C with extrac­tion buffer, 2% (v/v) Tri­ ton X100 was cen­tri­fuged at 20,000g for 20 min, and PMase puri­ fied by Ni-che­late chro­ma­tog­ra­phy. Prep­a­ra­tion of Physa­rum crude mem­brane sus­pen­sion. Plas­mo­ dia (grown for 2d) har­vested on a nylon sieve was washed three times with 25 mL por­tions of fresh cul­ture medium. Por­tions of 2 g plas­mo­dia were lysed at 4 °C dur­ing 8–10 strokes in a Do­un­ce homog­e­niser sus­pended in 3 mL ice-cold extrac­tion buffer (50 mM Tris–HCl, pH 7.5, 50 mM EGTA, 14 mM 2-mercap­toethanol, 10 mM MgCl2, 300 mM NaCl, pro­te­ase inhib­it­ or cock­tail, and 20% glyc­erol).

After pel­let­ing at 1000g for 15 min at 4 °C, post nuclear super­na­tants were again pel­leted at 20,000g for 20 min yield­ing the mem­brane and the cyto­plas­mic frac­tions. Mem­branes were resus­pended in 2 mL extrac­tion buffer on ice. Plasma mem­branes were puri­fied by iso­pyc­nic sucrose gra­di­ent cen­tri­fu­ga­tion [18]. Frac­tions start­ing from the bot­tom were assayed for PMase and alka­line phos­pha­tase (plasma mem­brane marker) activ­i­ties. Puri­fi­ca­tion of recombinant pro­tein. Sol­u­ble cell extract was loaded on a 1 ml Ni2+-charged Hi­sTrap FF col­umn (4 °C, Äkta Prime Sys­tem, GE Health­care, Munich, Ger­many) and washed with 100 mM Tris–HCl (pH 7.5), 300 mM KCl, 10 mM imid­az­ole, and PMase eluted with 100 mL lin­ear 10 mM–500 mM imid­az­ole gra­di­ ent between 50 mM and 150 mM imid­az­ole. After dial­y­sis against 50 mM Tris–HCl (pH 7.5), the puri­fied pro­tein was con­cen­trated in a Centri­con (Bille­rica, MA, USA) (15 mL). PMase acti­va­tion, enzyme assays and other meth­ods. Recombinant PMase was acti­vated in a mix­ture of 0.5 mL acti­va­tion buffer, 20–500 nM puri­fied recombinant PMase, 2% Tri­ton X100, 2 mM MgATP, and 50 lL puri­fied mem­branes (sus­pen­sion of mem­branes from 2 g Physa­rum plas­mo­dia) at 20 °C for var­i­ous times. After 20 min cen­tri­fu­ga­tion at 20,000g, 20 lL ali­quots were assayed for PMase activ­ity and/or ana­lyzed by SDS–PAGE—Western blot­ting (PMase anti­body). Back­ground activ­ity of mem­branes was mea­ sured in absence of recombinant PMase-pro­tein and sub­tracted from total activ­ity. The size of the acti­va­tion reac­tion mix­ture could be eas­ily up­scaled. PMLA and activ­ity of PMase were mea­sured as described [12]. PMase activ­ity refers to A340-units £ h¡1 of NADH in the stan­ dard (malate dehy­dro­ge­nase) assay equiv­a­lent to the amount of l-malate. Puri­fied PMase, PMase-His6-tag, PMase-His10-tag, PMase-Streptag, and PMase(devoid of sig­nal pep­tide) were quan­ti­tated using the spe­cific ab­sor­ben­cies 0.1%A280 [cm2 mg¡1] of 1.513, 1.497, 1.486, 1.544, and 1.555. SDS–PAGE, Western blot­ting with lumi­nes­cence detec­tion fol­lowed a man­u­fac­turer’s pro­to­col http://www.en­er­ gene.com/prod­ucts.htm#NOWA. For incor­po­ra­tion of 32P, mix­tures of 100 lL mem­branes in acti­va­tion buffer with 5 lCi of either [a-32P]ATP (3000 Ci/mmol, Amersham) or [c-32P]ATP (5000 Ci/mmol) were incu­bated 15 min at 25 °C and ana­lyzed by SDS–10%PAGE and auto­ra­di­og­ra­phy. Results and dis­cus­sion Syn­the­ses of recombinant PMAse-pro­tein First tests of recombinant syn­the­sis were car­ried out with E. coli. PMase-pro­tein expressed in E. coli NEB T7 RP and NEB T7 Rosetta, was detected in pel­lets and super­na­tants (Fig. 1). By Western blot­ting with PMase-spe­cific anti­se­rum (Fig. 1A) sev­eral pro­tein bands and by blot­ting with anti-His-tag anti­body a sin­gle pro­tein (Fig. 1B) revealed a poly­pep­tide of 75 ± 2 kDa degraded from the C-ter­mi­nal His-tag. The pro­tein was puri­fied from the sol­u­ble frac­tion (2 mg from 5 L induc­tion medium, OD600 = 1.0), and was enzy­mat­i­cally inac­tive. In prin­ci­ple, recombinant syn­ the­sis was fea­si­ble, but because of frag­men­ta­tion this route was not fol­lowed. Sac­cha­ro­my­ces ce­re­vi­si­ae strains W303 and c13 were use­ful. The degree of frag­men­ta­tion was accept­able in strain c13, used fur­ther on. His6-tag was replaced by His10-tag or Strep-tag to reduce cop­u­ ri­fi­ca­tion of yeast pro­teins (Fig. 1C and D). PMase-pro­tein was con­ tained in par­tic­u­late frac­tions and sol­u­bi­lized on stand­ing. After extrac­tion with 2% Tri­ton X100 (lane MS in Fig. 1F), sol­u­ble PMsepro­tein was puri­fied by Ni-che­late chro­ma­tog­ra­phy (3.5 mg from 5 L induc­tion medium, OD600 = 1). The molec­u­lar mass 92 ± 2 kDa was the same for pellet and super­na­tant and higher than that of E. coli recombinant pro­tein (75 ± 2 kDa) but less than 97 ± 2 kDa for P.



W. Mu­el­ler et al. / Biochemical and Biophysical Research Communications 377 (2008) 735–740

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Fig. 1. Recombinant expres­sion of PMase-pro­tein in Esch­e­richia coli and yeast. (A) Expres­sion of PMase in E. coli NEB T7 RP and NEB T7 Rosetta after induc­tion with 0.1 mM IPTG. Lane P, pellet; lane S, super­na­tant. Western blot (SDS–12.5%PAGE) with anti-PMase anti­body. (B) Same as a, but detec­tion with anti-His6 anti­body. (C) Puri­fi­ca­tion of Sac­cha­ro­my­ces ce­re­vi­si­ae c13 recombinant PMase-His10 by Ni-che­late affin­ity chro­ma­tog­ra­phy. Lane C, crude cell extract; lane X, col­umn break-through, lanes 13–23, peak elu­tion; lane St, molec­u­lar mass stan­dards (SDS–8%PAGE, Coomassie Bril­lant Blue stain­ing). (D) Puri­fi­ca­tion of yeast recombinant PMA­ase-pro­tein-Strep by StrepT­ac­tin chro­ ma­tog­ra­phy; lane W2 and W5, col­umn wash; lanes E1–E8 peak elu­tion (SDS–8%PAGE, sil­ver stain­ing). (E) Yeast recombinant PMase trun­cated from sig­nal pep­tide. Lane K, non-trun­cated PMase; lane MP, trun­cated PMase in the mem­brane pellet; lane S, trun­cated PMase in the super­na­tant. (F) Yeast was unable to secrete recombinant PMasepro­tein as shown in lane K for con­cen­trated sam­ple of cul­ture broth after ammo­nium sul­fate pre­cip­i­ta­tion. Yeast recombinant PMase is shown in frac­tion­ated cell extracts: Lane C, yeast crude extract. lane P1, pellet after low speed cen­tri­fu­ga­tion; lane S, super­na­tant after a sec­ond, high speed cen­tri­fu­ga­tion in the absence of Tri­ton X100, and the cor­re­spond­ing pellet in lane P2. This pellet was extracted in the pres­ence of 2% Tri­ton X100 in lane MS. Western blots (SDS–8%PAGE, PMase anti­body) were prepared with 5 lL of 10-fold diluted sam­ples.

poly­ceph­alum. Molec­u­lar mass was 75 kDa when the leader pep­tide was trun­cated (yeast PMase), and iden­ti­cal with PMase expressed by E. coli (Fig. 1A, B and E, lanes S, MP). Although recombinant PMase was gly­cos­yl­ated, it was not secreted into the cul­ture broth (no detec­tion even after con­cen­tra­tion by 65% ammo­nium sul­fate pre­cip­i­ta­tion, lane K, Fig. 1F). The recombinant PMase was not enzy­mat­ic­ ally active.

In search of PMase acti­va­tion Dis­tri­bu­tion of endog­e­nous PMase-pro­tein As with E. coli and yeast, PMase of P. poly­ceph­alum was con­ tained in the par­tic­u­late and its release dur­ing stand­ing was increased in the pres­ence of 2% Tri­ton X100 (Fig. 2). At var­i­ance

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Fig. 2. PMase acti­va­tion by sol­u­bi­li­za­tion. (A) PMase activ­ity in frac­tions of plasma mem­branes puri­fied by iso­pyc­nic 30–65% sucrose gra­di­ent cen­tri­fu­ga­tion [18]. Activ­i­ties of PMase and inor­ganic phos­pha­tase (marker for plasma mem­branes) frac­tion­ate together. (B) Sol­u­bi­lized PMase-pro­tein by Western blot­ting (anti-PMase anti­body) appeared on stand­ing of plasma mem­branes in the super­na­tant together with hydro­lase activ­ity. (C) The same exper­im ­ ent as in fig­ure b in the pres­ence of 2% Tri­ton X100. (D and E) Spon­ta­ne­ous frag­men­ta­tion of PMase in extracts and cul­ture broth of 2 and 3 days old plas­mo­dia. Lane B, cul­ture broth; lane P1, pellet con­tain­ing nuclei and cell frag­ments; lane P2, mem­brane pellet; lane S1, super­na­tant of mem­brane pellet; lane E, crude extract. (F) PMase activ­ity appeared in the super­na­tants of panel B (no Tri­ton X100) and C (2% Tri­ton X100). Means and stan­dard devi­a­tions refer to exper­i­ments in trip­li­cates.

with yeast, released pro­tein was enzy­mat­i­cally active, and the cul­ture broth of Physa­rum con­tained enzy­mat­ic­ ally active PMase [1,12]. When Physa­rum plasma mem­brane was puri­fied by iso­ pyc­nic cen­tri­fu­ga­tion, PMase frac­tion­ated together with inor­ ganic phos­pha­tase, a marker for plasma mem­branes (Fig. 2A). The puri­fied mem­brane frac­tion itself was devoid of hydro­lase activ­ity, how­ever, on stand­ing, PMase activ­ity appeared together with sol­u­bi­lized PMase-pro­tein (Fig. 2B and F), and increased in the pres­ence of 2% Tri­ton X100 (Fig. 2B, C, and F). The results sug­gested that PMase-acti­vase was a con­stit­ue ­ nt of the plasma mem­brane. The enzy­matic nature of Physa­rum acti­vase Because Physa­rum PMAse-pro­tein was rap­idly cleaved under a vari­ety of con­di­tions (Fig. 2D and E), acti­va­tion was at first con­ sid­ered the result of a pep­tide cleav­ing acti­vase [12], but then was

con­sid­ered unlikely after find­ing the acti­va­tion resis­tant to pro­te­ ase inhib­i­tors (data not shown). Dam­age of plasma mem­branes is known to inhibit in vivo syn­ the­sis of PMLA by pro­tein phos­phor­y­la­tion [19]. On that basis, the idea was born that phos­phor­y­la­tion acti­vated PMLA deg­ra­da­tion in a com­ple­men­tary fash­ion. To test whether a kinase was acti­ vase, puri­fied Physa­rum plasma mem­branes were incu­bated with 2 mM MgATP and the super­na­tant assayed for PMase activ­ity (Fig. 3A and B). Hydro­lase activ­ity appeared sev­eral-fold over the back­ ground by the mem­branes alone (Fig. 3A) and Tri­ton X100 had an acti­vat­ing effect (Fig. 3B). The results in Fig. 3A inset ruled out that MgATP increased acti­va­tion via sol­u­bi­li­za­tion. The acti­va­tion was inhib­ited by Tyr­pho­stin-47, a pro­tein tyro­sine-kinase inhib­i­tor, or by tyro­sine-spe­cific pro­tein-phos­pha­tase. The results sug­gested acti­va­tion by a mem­brane bound pro­tein tyro­sine kinase. Hydro­lase activ­ity unfolded only after dis­so­ci­a­ tion of acti­vated PMase from the mem­brane. Back­ground activ­ity

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W. Mu­el­ler et al. / Biochemical and Biophysical Research Communications 377 (2008) 735–740

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50 0 PM nM re As co e-p mb rot . .

25 0 PM nM re As co e-p mb rot . .

Ze r PM o rec 50 Ase omb 0n . M prot BS A. 50 n PM M re As com e-p rot b. .

Ze r PM o rec As om e-p b. rot .

Activation time (h)

Fig. 3. PMase acti­va­tion by phos­phor­y­la­tion. Endog­e­nous and recombinant PMase were acti­vated in the pres­ence of sus­pended Physa­rum mem­branes. Ingre­di­ents were 2 mM MgATP, and as indi­cated: 2% Tri­ton X100, 10 lM Tyr­pho­stin-47, 40 lg/mL Tyr-phos­pha­tase. (A) Emerg­ing activ­ity of endog­e­nous PMase on stand­ing of plasma mem­branes prepared by iso­pyc­nic sucrose gra­di­ent cen­tri­fu­ga­tion. Fig­ure inset shows sol­u­bi­lized PMase in the pres­ence of Tri­ton X100 or MgATP (Western with anti-PMase anti­body). (B) Emerg­ing PMase activ­ity on stand­ing of crude mem­branes in the pres­ence of Tri­ton X100. (C) Emerg­ing PMase activ­ity of yeast recombinant PMase on top of endog­e­nous PMase (back­ground acti­va­tion) dur­ing stand­ing in the pres­ence of added Physa­rum mem­branes. Back­ground acti­va­tion is shown in the absence of recombinant PMase with or with­out bovine serum albu­min (BSA). Tri­ton X100 was pres­ent except where indi­cated. Aver­age val­ues and stan­dard devi­a­tions refer to mea­sure­ments in trip­li­cates. Fig­ure inset shows 32P-incor­po­ra­tion into pro­teins of puri­fied plasma mem­branes after reac­tion with [a-32P]ATP (lane a) or [c-32P]ATP (lane b).

by sol­u­bi­li­za­tion was explained by in loco acti­va­tion of mem­brane bound PMase-pro­tein dur­ing par­tic­u­late prep­a­ra­tion. Acti­va­tion of recombinant PMA­ase The results obtained for Physa­rum PMAse sug­gested the method for recombinant pro­tein acti­va­tion. This was born out in Fig. 3C show­ing activ­ity pro­por­tional to added amounts of puri­ fied recombinant PMAse-pro­tein that could not be substi­tuted by bovine serum albu­min (BSA). The net activ­ity for 500 nM added recombinant PMAse-pro­tein cor­re­sponded to a spe­cific hydro­lase activ­ity of 560 U/mg com­par­ing with 540 U/mg activ­ity for PMAse

iso­lated from cul­ture broth [1]. Reac­tion of Physa­rum mem­branes with [c-32P]MgATP indi­cated phos­phor­y­la­tion of a 97 kDa pro­tein (Fig. 3C, inset), while this was not observed with [a-32P]ATP rul­ing out ade­ny­la­tion. Recombinant expres­sion and con­trolled PMase acti­va­tion allow prep­a­ra­tion of active enzyme for bio­tech­ni­cal appli­ca­tion such as na­no­con­ju­gate tai­lor­ing and pro­vide exper­i­men­tal means to val­i­date sup­posed in vivo func­tions of PMase and PMLA thereby advanc­ing knowl­edge about the phys­i­ol­ogy of plas­mo­dia. ­Emerg­ing is the role of pro­tein tyro­sine kinase in reg­u­lat­ing syn­the­sis and deg­ra­da­tion of PMLA under the con­trol of plasma mem­brane dam­ age [19]. In this con­text, acti­va­tion of Physa­rum trans­glu­ta­min­ase

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is men­tioned that involves Ca2+ [20]. The sec­ond mes­sen­ger could down­regu­late PMLA via acti­vat­ing tyro­sine pro­tein kinase(s). Acknowl­edg­ment The inter­est and sup­port by Dr. Rein­hard Sterner, Insti­tut fuer Bio­phys­ik und Phys­i­ka­li­sche Bi­oche­mie der Uni­ver­si­taet Re­gens­ burg, Ger­many, is greatly acknowl­edged. Ref­er­ences [1] C. Korh­err, M. Roth, E. Hol­ler, Poly(b-l-malate) hydro­lase from plas­mo­dia of Physa­rum poly­ceph­alum, Can. J. Micro­biol. 41 (1995) 192–199. [2] H. Fischer, S. Erd­mann, E. Hol­ler, An unusual pol­y­an­ion from Physa­rum poly­ ceph­alum that inhib­its homol­o­gous DNA poly­mer­ase alpha in vitro, Bio­chem­ is­try 28 (1989) 5219–5226. [3] T. Göt­tler, E. Hol­ler, Screen­ing for beta-poly(l-malate) bind­ing pro­teins by affin­ity chro­ma­tog­ra­phy, Bio­chem. Bio­phys. Res. Com­mun. 341 (2006) 1119– 1127. [4] K. Rath­ber­ger, H. Re­is­ner, B. Willi­bald, H.-P. Mo­li­to­ris, E. Hol­ler, Com­par­at­ ive syn­the­sis and hydro­lytic deg­ra­da­tion of b-poly(l-malate) by myx­o­my­ce­tes and fungi, Mycol. Res. 103 (1997) 513–520. [5] B.-S. Lee, M. Vert, E. Hol­ler, Water-sol­u­ble ali­phatic poly­es­ters: poly(malic acid)s, in: Y. Doi A. Stein­bü­chel (Eds.), Bio­poly­mers, Wiley-VCH, Wein­heim, 2002, pp. 75–103. [6] B.-S. Lee, M. Fuj­it­a, N.M. Khaz­en ­ on, K.A. Waw­row­sky, S. Wachs­mann-Hog­iu, D.I. Fark­ash, K.L. Black, J.Y. Lju­bim­ova, E. Hol­ler, Po­ly­ce­fin, a new pro­to­type of mul­ti­func­tional na­no­con­ju­gate based on poly(beta-l-malic acid) for drug deliv­ery, Bio­con­jug. Chem. 17 (2006) 317–326. [7] J.Y. Lju­bim­ova, M. Fuj­it­a, A.V. Lju­bi­mov, V.P. Torchi­lin, K.L. Black, E. Hol­ler, Poly(malic acid) na­no­con­ju­gates con­tain­ing var­io ­ us anti­bod­ies and oli­go­ nu­cle­o­tides for mult­itar­get­ing drug deliv­ery, Nano­med­i­cine 3 (2008) 248– 265. [8] A. Schmidt, C. Wind­i­sch, E. Hol­ler, Nuclear accu­mu­la­tion and homeo­sta­sis of the unusual poly­mer b-poly(l-malate) in plas­mo­dia of Physa­rum poly­ceph­ alum, Eur. J. Cell Biol. 70 (1995) 373–380.

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