PETER J. WHITE, DAVID M. MOUSDALE and. JOHN R. COGGINS. Dcpurtment of' BiochcJmistry. University of' Glasgow. GI2 RQQ. Scotland, U.K.. Chorismate ...
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PETER J. WHITE, D A V I D M. M O U S D A L E and J O H N R. C O G G I N S Dcpurtment of' BiochcJmistry. University of' Glasgow. G I 2 RQQ. Scotland, U . K . Chorismate synthase (5-enolpyruvylshikimate 3-phosphate phospho-lyase; EC 4.6. I .4) is the seventh and final enzyme of the common pathway of aromatic biosynthesis (the shikimate pathway) which is found both in micro-organisms and in plants (Haslam. 1974; Weiss & Edwards. 1980). It catalyses the conversion of 5-enolpyruvylshikimate 3phosphate (EPSP) to chorismic acid, the key branch point intermediate in the biosynthesis of all the aromatic compounds synthesized via the shikimate pathway. Chorismate synthase is the least well understood and least characterized of all the shikimate pathway enzymes. It has an unusual reaction mechanism (Floss, i't ul.. 1972; Ganem, 1978) and in some species the enzyme is unstable and difficult to assay. The chief difficulty is the complex, and so far incompletely defined, requirement for reduced flavin. Although the overall reaction involves n o net change in oxidation state, the enzyme seems to be dependent for activity on the presence of a reduced-flavin-generating system ( a diaphorase activity). The enzyme has been characterized from three microbial sources (Escherichiu c d i , Morell t t ul.. 1967; Nrurosporu u u s u , Welch et al., 1974; Boocock, 1983; Bucillis suhti1i.s. Hasan & Nester, 1978) and recently from one plant source (Mousdale & Coggins, 1986). The microbial enzymes, although catalysing the same reaction, differ markedly in their cofactor requirements, quaternary structure and sensitivity to molecular oxygen. Both the N . crussa and B. suhtilis chorismate synthases can be readily assayed under aerobic conditions with F M N and N A D P H as cofactors. In contrast, the chorismate synthase partially purified from E. c d i could only be assayed under strictly anaerobic conditions in the presence of chemically or enzymically reduced flavin or if the enzyme were treated with dithionite or H,/platinum (Morell et ul., 1967). Pea seedling chorismate synthase must also be assayed under strictly anaerobic conditions (Mousdale & Coggins, 1986). Detailed studies of the E. coli enzyme have been hindered by its low abundance in wild-type strains and its acute sensitivity to molecular oxygen. In an attempt t o understand the mechanism of action of chorismate synthase the E. coli gene (uroC') which encodes chorismate synthase has been cloned and overexpressed (Millar et al., 1986). To quantify the level of overexpression and to facilitate the purification of the overproduced enzyme a rapid and simple anaerobic
assay procedure for the enzyme was required. The assay procedures employed in earlier work on the E. coli enzyme relied on measuring the phosphate produced in the reaction, which is difficult to d o reproducibly in crude extracts, o r on the availability of coupling enzymes (Morell ct d..1967). We have developed a new procedure involving the direct measurement of the chorismate formed in the reaction after separation of the reaction products by h.p.1.c. on a cationexchange column. This can be simply achieved because
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Abbreviation used: EPSP. 5-enolpyruvylshikimate 3-phosphate.
Fig. 1. Elution proJile of a typical chorisrnatesynthase assay mi.uture ,from a Biorad Aminex HPX-87H cation-exchange column The standard 1 ml assay contains: 50 mM-potassium phosphate buffer pH 7.0. ~ O ~ M - E P SIOpm-FAD. P. 2 mM-sodium dithionite and the enzyme sample. To initiate the assay an aliquot of sodium dithionite is added to a cocktail containing all the other components including enzyme. The reaction vessel is stoppered and incubated in a waterbath at 25 C. Aliquots (20 j t l ) of the reaction mixture are removed at various time points and applied immediately to a Biorad Aminex HPX-87H column (column dimensions l00mm x 7.8mm; flow rate I.Oml/min; solvent 20 mM-H,SO,). Chorismic acid elutes from the column at 9min and is detected at 215nm. The amount of chorismic acid is determined by peak integration and comparison with standard samples of known concentration.
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I45
chorismatc is significantly retarded on Aminex-type strongcation-exchange columns (Mousdale & Coggins, 1985) and can therefore be readily separated from the substrate EPSP iind the other components required in the assay. In the new assay procedure anaerobic conditions are generated via reduced flavin which is produced by the action of sodium dithionite on F A D . The assays are carried out in 1 ml spectrophotometer cuvettes sealed with 'Subaseal' stoppers. At various time intervals samples are removed with a syringe, injected directly into an h.p.1.c. apparatus and the chorismic acid separated and analysed. A typical column elution profile is shown in Fig. 1 and details of the assay conditions are given in the legend. The assay was found to be reproducible and linear over a IOmin time period. Each chorismic acid analysis took approx. I5 min. This assay technique has facilitated the development of a purification procedure for chorismate synthase from E. c d i strain AB2849/pGM602 (P. J . White, G. Millar & J. R. Coggins. unpublished work). The generation of anaerobic conditions requires no specialized equipment and it should
now be possible to make more rapid progress with the investigation of the mechanism of this interesting enzyme. Boocock. M. R. (19x3) Ph.D. Thesis. University of Glasgow Floss. H . G.. Ondcrka. D. K . &Carroll. M. (1972) J . Biol. Chcn7. 247. 736 744 Ganem, B. (197X) Tt,rrtrlichui L o / . 34. 3353 3383 Hasan. N . & Nester, E. W. (1978) J . Btol. Chc~ni.253. 4993 4998 Haslam. E. ( 1974) Tlu, Shikiniurc, P u / h ~ ~ uEutterworths. y. London Morell. H.. Clark. M. J.. Knowles. P. F. & Sprinson. D. B. (1967) J . B i d . C'l7cvi. 242. 82 90 Millar. G . , Anton. I . A.. Mousdale. D . M.. White. P. J. & Coggins. J. R. ( 1986) Biod7cn7. Sot,. Tru)i.s. 14. 262 263 Mousdale. D. M. & Coggins. J . R. (1985) J . Chromuiogr. 329. 268 272 Mousdale. D. M . & Coggins, J. R. (1986) FEES Lei/. 205, 328-332 Welch. G. R., Cole, K . W. & Gaertner. F. H. (1974) Arch. Biochem. Biopltys. 165. 505 5 I 8 Weiss. U. & Edwards. J. H. (1980) Thc Biosynrhesis of' Aromulic, Conipounds. J. Wilcy, New York Received 15 July 19x6
Neurotoxin-stimulated sodium transport in insect central nervous system synaptosomes A. K. DWIVEDY A . F. R . C . Unit o f ' Insect New-ophysiology and Phtirt?itic,olo~gj. , Dcpir t m m t of' Zoology, Uniivrsitj. ()f' Ciitiibridgc. Downing Street. C'triiihrii/cgc~ CB2 3 E J . U . K . Sodium channels are voltage-gated ionic channels essential for the generation of action potential in various excitable membranes. The pharmacological agents used as functional probes of sodium channels suggest distinct receptor sites for various toxins defined predominantly as sodium flux inhibitors (tetrodotoxin, saxitoxin), sodium channels activators (veratridine, aconitine, batrachotoxin), and inactivation inhibitors (sea anemone toxin) (Catterall, 1980). The responses of specific toxins to sodium channels differ considerably in rat, mouse brain synaptosomes, mouse neuroblastoma cells (Ghiasuddin & Soderlund, 1984) and rat brain neurons (Couraud et a f . , 1986). The present investigation describes appropriate conditions for measuring sodium Abbreviation used: CNS. central nervous system
Table I . Compurison
of'
channel-dependent '2Na+ flux in the insect (Periplaneta unwricuna) central nervous system (CNS) synaptosomes. The results suggest that veratridine and aconitine both produced a n increase in the rate of influx of N a + into CNShomogenate and isolated synaptosomal preparation. Tetradotoxin antagonized the stimulatory effect of veratridine o n the influx of "Na' ( I < , , , 2 3 . 5 ~ while ) the polypeptide neurotoxin (Anemonia sulcata-ll) potentiated the stimulatory effect of veratridine o n the uptake of "Na+ ( K , , , 2.0 nM). --NaCI (carrier free, lOOmCi/mg) was purchased from Amersham International U.K. Veratridine, aconitine and other chemicals were purchased from Sigma Chemicals, U.K. Synaptosomes were prepared from insect C N S according to the method previously described (Dwivedy, 1985). Aliquots ( 10-20 pg protein) of synaptosomal suspension were incubated a t 21 C for 3 0 s with various toxins in a sodium incubation buffer (Ghiasuddin & Soderlund, 1984). Uptake of "Na+ was measured by rapid filtration technique (Whatman C filter, 0.45 p m pore) followed by liquid scintillation counting. The protein content of the ?,
apparent afiniticv, K,, of'neuroto.uin c:fii.t.s on "Nu $,
uptake
K O , is the concentration giving a half-maximal activation. Data arc fitted to a modified form of the Michaelis-Menten equation (Catterall. 1980). n.d.. not determined.
Preparation
Activators . . .
Mouse brain synaptosomes* Rat brain synaptosomes* Mouse neurobalstoma cells* Cultured mouse neuronal cellst ?-day fetal brain 9 12-day fetal brain Insect CNS (present study) CNS homoyenate Synaptosomes:
Batrachotoxin
Veratrdine
Aconitine
0.49 0.5 0.7
34.5 13.0 29.0
19.6 14.0 3.6
6.0 0.3
90 X.6
n.d. n.d.
9.5 3.09
15.0 7.6
0.25 0.20
*From Ghiasuddin & Soderlund (1984) ?From Couraud el ul., (1986) :Initial rate of "Nat uptake in absence of activator was 57 nmol/30 s perjmg of protein. SVeratridine activation of 22Na+ influx was inhibited by tetradotoxin (15(l. 23.5 nM) and potentiated by Anemonia sulcata-I1 ( K o 5 ,2 . 0 n ~ ) .
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