An assay for argininosuccinate synthetase in Neurospor

Fairley, J. L.

Wampler, D. E.

Fungal Genetics Reports

English

D. E. Wampler

J. L. Fairley

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An assay for argininosuccinate synthetase in Neurospora

Kansas State University

Fungal Genetics Reports Volume 8 Article 29 An assay for argininosuccinate synthetase in Neurospora D. E. Wampler J. L. Fairley Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Wampler, D. E., and J.L. Fairley (1965) "An assay for argininosuccinate synthetase in Neurospora," Fungal Genetics Reports: Vol. 8, Article 29. https://doi.org/10.4148/1941-4765.2136 This Technical Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. An assay for argininosuccinate synthetase in Neurospora Abstract An assay for argininosuccinate synthetase in Neurospora This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol8/iss1/29 Taylor, C. W. A more efficient procedure for scoring A technique for scoring abermtionr  anwng  isolates  by crossingmating type and  aberrations. them with standard fluffy strains (fl (P605) a and UPA)  wetsdescribed by Perkins et al.  (1962 Can. J. Gene+. Cytol.  4:187).Strains to be tested were spotted on plater containing fertile fluffy testers,  and aberratlonr scored by the frequency of white sporesr-shot to the lid of the plate. It has now  been found to be technically advantageous for large numbers of sex  tests  and  far scoringaberrations to make the crosses  in 3-inch tuber rather than on plater.The advantages of tubes over plater are these. The tests can be done by relatively unskilled help without the possibility ofrcotter either in the formation of Perithecia or in ascorpore  shooting. There is no problem of spores moving in the condensatethat forms on the lid of a plate. Any error is thus eliminated in determining which spores  came from a particular i-late. Pori-tive  tests can be used for progeny testing, if necersary.Tubes are inoculated using a suspension of fluffy mycelia prepared as  fallows: the two fluffy strains  are  grown 4 days  at 34OCin 300 ml flasks contoining  50 ml of o liquid glycerol complete medium (a variation of Medium 2 described by Tatum  c 4  .,1950 Am. J. Botany 37:38)  2nd  a 15 cm filter paper cone (for greater oeriol growing surface). Several flasks can be preparedand  grown up ot one time and  then refrigerated until needed (good for severa!  weeks). A dense mycelial  suspension is mode byadding about 50 ml sterile water to each flask and vigorously shaking, first by hand and then with (I vortex mixer. Three-inchtubes of SC ogor  (Synthetic Cross Medium, Westergoard  and Mitchell 1947 Am, J. Botany 34:573)  ore inoculated with o dropof the myceliol  suspension, using o wide-bore Pasteur pipette with a squeeze bulb. SC is color-coded with Schilling food colorbefore dispensing, to identify the +wo  fluffy mating types and minimize mix-ups.Tubes inoculated  with the flufQ  +&ens tire  incubated at 25°C until protoperithecio  are formed. They are then ready for fer-tilization, or they may be stored a+  5’C  and used up to one week later. Sex +esh  ore rcorable after four days  at 25°C andaberrations (fertility and frequency of aborted spores)  can be determined within 12-14 days after crossing  by examining asco-spores  shot  to the wall  of the +ube.  Tests  are  routinely made against  testers  of both  mating types  to reveal  false negatives,  infer-tility, or birexucllity.  - - - Department of Biological Sciences, Stanford University, Stanford, California.Troo, M. U. and  M.  W. Smith. Concentration of This is a technique we have developed for use in the purificationchromotographic  effluent for gel electrophoresis. of dehydrogenores  from Neumrpxa  but it may  hove other appli-cations. In the purification of enzyme extracts the fractions ofeffluent collected from the chromatographic column ore much too dilute for direct application to supporting material for gel elec-trophoreris. A simple method of concentrating the effluent was  needed to allow detection of ba?ds  of protein or isoenzymes  afterelectrophoresir.  This method is bared on the technique first described by Horowitz and Fling ( 1962 NNX2:  19) who placeddilute wlutions  of tyrosinare  in dialysis bags  +a dialyze against  solid  wcroze.  We reversed the positions of the sucrose and thesample. Sucrose growler  ore  placed  in a dialysis bag of large diameter. The top surface of the bog is flattened so  that a row offilter paper  pieces can be placed in close contact with the surface. The bog is chilled and cold fractions of effluent are pipettedonto the surface until a layer of liquid is formed on top of each piece. Water and electrolytes ore obrorbed  into the sucrose be-low and  in on hour or so a second application, if necerrory,  can  be made or the filter paper piecer can now be inserted into starchor ocrylomide  gel for electrophorerir. This method allows  (I large number of samples to be concentrated simultaneously and  re-quires very little expenditure of equipment or material.  The pipetted amounts of effluent also give a control of the amount ofprotein or enzymes to be subjected to electrophorerir. - - - Pediatric Loborotory,  University of Michigan Medical Center, AnnArbor, Michigan.Wampler,  D. E. clnd  J. L. Fairley.  An array  for During on investigation of arginine  synthesis in N. crc~ssu,  itargininoruccinclte synthetare in Neurasporo. become necessary for us to assay for orgininoruc~a~thetase.A method bored on the disappearance of citrulline from reactionmixtures has been described (Newmeyer 1962 J. Gen. Microbial.  28:215),  but little information was presented concerning con-ditions for optical activity. The present report describes a modified, more sensitive procedure developed in this  l&orator/.Culture conditions and the method of preparing acetone powders have appeared elsewhere (Fairley  and Wompler 1964 Arch.Biochem.  Biaphyr. 106:  153). The acetone powder is added to 0.01 M phosphate buffer pH  7.4 (I g/9 ml), homogenized in CIServ.11  Chnnimixer  and  centrifuged for 15 min ot 23,000 x g. The residue is resuspended in (I small  volume,of  buffer and recen-trifuged.  The combined rupernotclnt  liquids (containing IO-15 mg protein/ml) are  added to CI  column of Sephodex  G-25 (a 2.2x 25 cm column is adequate for CI  15-ml  sample) which has been equilibrated with 0.01 M phosphate buffer, pH  7.4. The typeof buffer is not critical and  any pH between 7.0 and 8.2 is satisfactory. Protein is elated with the same  buffer and tubes con-taining more than  2 mg protein per ml, as  meawred  in the Biuret  reaction, are saved. Preparations which are not parsed throughSephadex  (or dialyzed for several  hours) do not exhibit activity which is lineor with enzyme concentration.The Sephodex-treated enzyme is osryed  in (I Z-ml reaction mixture which contains: 38  pmoles  potassium  aspartote, pH  8.0;22 pmoles  MgSO4; I .5 pmoler L-citrulline;  2.0 pmoler ATP; 20 pmoler 3-phorplwglyceric  acid and 100  pmoler Tris-chloridebuffer, pH  8.0. In this crude stage cl+ least 0.5 mg of protein must  be used for appreciable reaction to occur. The mixture isincubated ot 37’C  for IO minutes and  the reaction is stopped with I ml of I N perchloric acid.  Three ml of water is added(final volume 6 ml) and  the mixture is cen+rifuged. A one-ml sample of the wpernatant  is assayed for citrulline by the methodof Gerhord and  Pardee  (1962 J. Biol.  Chem. 237: 891). Th re eaction can  be run with 0.5 pmoles  citrulline and arrayed with-out diluting (final volume  3 ml) but these conditions result  in slightly lower apparent activity.The reaction is linear with respect to protein concentration within the range  used  cmd  with respect to time up to 10 minutes(Fig. 1 ). One of the reagents, ATP, is both  a substrate and an  inhibitor for the ieoction as  shown in Fig. I.  Fig. 2 shorn therelationship between activity and pH, using  three buffers. There is a fairly broad pH  optimum centered near pH  7.8.The enzyme is quite stable, retaining at  least  90% of its  original activity after storage at 3X  for three days. It can also bedialyzed for up to 16 hours  without detectable loss of activity. - - - Department of Biochemistry, Michigan State University,East Lansing Michigan. ( Resent addreu  of DEW; Dartmouth Medical School, Hanover, New Hampshire ).

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An assay for argininosuccinate synthetase in Neurospora

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